Isolated human secreted proteins, nucleic acid molecules encoding human secreted proteins, and uses thereof

ABSTRACT

The present invention provides amino acid sequences of peptides that are encoded by genes within the human genome, the secreted peptides of the present invention. The present invention specifically provides isolated peptide and nucleic acid molecules, methods of identifying orthologs and paralogs of the secreted peptides, and methods of identifying modulators of the secreted peptides.

FIELD OF THE INVENTION

[0001] The present invention is in the field of secreted proteins thatare related to the lactate dehydrogenase secreted protein subfamily,recombinant DNA molecules, and protein production. The present inventionspecifically provides novel peptides and proteins that effect proteinphosphorylation and nucleic acid molecules encoding such peptide andprotein molecules, all of which are useful in the development of humantherapeutics and diagnostic compositions and methods.

BACKGROUND OF THE INVENTION

[0002] Secreted Proteins

[0003] Many human proteins serve as pharmaceutically active compounds.Several classes of human proteins that serve as such active compoundsinclude hormones, cytokines, cell growth factors, and celldifferentiation factors. Most proteins that can be used as apharmaceutically active compound fall within the family of secretedproteins. It is, therefore, important in developing new pharmaceuticalcompounds to identify secreted proteins that can be tested for activityin a variety of animal models. The present invention advances the stateof the art by providing many novel human secreted proteins.

[0004] Secreted proteins are generally produced within cells at roughendoplasmic reticulum, are then exported to the golgi complex, and thenmove to secretory vesicles or granules, where they are secreted- to theexterior of the cell via exocytosis.

[0005] Secreted proteins are particularly useful as diagnostic markers.Many secreted proteins are found, and can easily be measured, in serum.For example, a ‘signal sequence trap’ technique can often be utilizedbecause many secreted proteins, such as certain secretory breast cancerproteins, contain a molecular signal sequence for cellular export.Additionally, antibodies against particular secreted serum proteins canserve as potential diagnostic agents, such as for diagnosing cancer.

[0006] Secreted proteins play a critical role in a wide array ofimportant biological processes in humans and have numerous utilities;several illustrative examples are discussed herein. For example,fibroblast secreted proteins participate in extracellular matrixformation. Extracellular matrix affects growth factor action, celladhesion, and cell growth. Structural and quantitative characteristicsof fibroblast secreted proteins are modified during the course ofcellular aging and such aging related modifications may lead toincreased inhibition of cell adhesion, inhibited cell stimulation bygrowth factors, and inhibited cell proliferative ability (Eleftheriou etal., Mutat Res 1991 March-November;256(2-6): 127-38).

[0007] The secreted form of amyloid beta/A4 protein precursor (APP)functions as a growth and/or differentiation factor. The secreted formof APP can stimulate neurite extension of cultured neuroblastoma cells,presumably through binding to a cell surface receptor and therebytriggering intracellular transduction mechanisms. (Roch et al., Ann N YAcad Sci 1993 Sep. 24;695:149-57). Secreted APPs modulate neuronalexcitability, counteract effects of glutamate on growth cone behaviors,and increase synaptic complexity. The prominent effects of secreted APPson synaptogenesis and neuronal survival suggest that secreted APPs playa major role in the process of natural cell death and, furthermore, mayplay a role in the development of a wide variety of neurologicaldisorders, such as stroke, epilepsy, and Alzheimer's disease (Mattson etal., Perspect Dev Neurobiol 1998; 5(4):337-52).

[0008] Breast cancer cells secrete a 52K estrogen-regulated protein (seeRochefort et al., Ann N Y Acad Sci 1986;464:190-201). This secretedprotein is therefore useful in breast cancer diagnosis.

[0009] Two secreted proteins released by platelets, platelet factor 4(PF4) and beta-thromboglobulin (betaTG), are accurate indicators ofplatelet involvement in hemostasis and thrombosis and assays thatmeasure these secreted proteins are useful for studying the pathogenesisand course of thromboembolic disorders (Kaplan, Adv Exp Med Biol1978;102:105-19).

[0010] Vascular endothelial growth factor (VEGF) is another example of anaturally secreted protein. VEGF binds to cell-surface heparan sulfates,is generated by hypoxic endothelial cells, reduces apoptosis, and bindsto high-affinity receptors that are up-regulated by hypoxia (Asahara etal., Semin Interv Cardiol 1996 September;1(3):225-32).

[0011] Many critical components of the immune system are secretedproteins, such as antibodies, and many important functions of the immunesystem are dependent upon the action of secreted proteins. For example,Saxon et al., Biochem Soc Trans 1997 May;25(2):383-7, discusses secretedIgE proteins.

[0012] For a further review of secreted proteins, see Nilsen-Hamilton etal., Cell Biol Int Rep 1982 September;6(9):815-36.

[0013] Lactate Dehydrogenase

[0014] Lactate dehydrogenase (LDH), sometimes referred to as lacticdehydrogenase, is the most clinically important dehydrogenase occurringin human serum. LDH is clinically important because serum level ofcertain isozymes reflect pathological conditions in particular tissues.Consequently, LDH is most often measured to evaluate the presence oftissue damage. LDH serves as an indicator suggestive of disturbances ofthe cellular integrity induced by pathological conditions. Since LDH isan enzyme present in essentially all major organ systems, serum LDHactivity is abnormal in a large number of disorders.

[0015] LDH is found in the cytoplasm of cells and catalyzes thefollowing reversible reaction for the interconversion of pyruvate andlactate: Pyruvate+NADH⇄Lactate+NAD

[0016] This bidirectional reaction can be monitoredspectrophotometrically by measuring either the increase in NADH at 340nm produced in the lactate-to-pyruvate reaction, or by measuring thedecrease in NADH at 340 nm produced in the pyruvate-to-lactate reaction.

[0017] Mammalian LDH exists as five tetrameric isozymes composed ofcombinations of two different polypeptide subunits with a molecularweight of 136,700±2,100 daltons per tetramer. These tetrameric isozymeseach differ in catalytic, physical, and immunological properties. Cahnet al., (1962) designated the two polypeptide subunits as H (heart) andM (muscle), which combine to form two pure types of isozymes, H4 and M4,and three hybrids, H3M, H2M2 and HM3. Type H4 is the most negativelycharged at pH 7 and appears nearest the anode upon zone electrophoresis.Subunit H predominates in heart muscle and facilitates the aerobicoxidation of pyruvate. The M subunit predominates in skeletal muscle andliver and is predominantly involved with anaerobic metabolism andpyruvate reduction.

[0018] Iodide inhibits LDH; p-mercuribenzoate also inhibits LDH, but ata slower rate. LDH is activated by a number of organic compounds thatstabilize the enzyme, such as dimethyl sulfoxide, ethanol, and methanol.Diethystilbestrol and several of its derivatives also stabilize theenzyme.

[0019] Normal total LDH levels are approximately 105 to 333 IU/L(international units per liter). Higher than normal LDH levels may beindicative of such conditions as cerebrovascular accident (e.g., CVA,stroke), myocardial infarction, hemolytic anemia (as well as other formsof anemia), hypotension, infectious mononucleosis, intestinal ischemiaand infarction, liver disease (e.g., hepatitis), muscle injury, musculardystrophy, neoplastic conditions, pancreatitis, and pulmonaryinfarction. If the total LDH level is elevated, specific serum LDHisoenzymes may be measured to increase the accuracy of diagnosis.

[0020] The most frequent use of LDH isoenzyme analysis is for thediagnosis of myocardial infarction and other myocardial diseases. SerumLDH levels are elevated in various myocardial diseases and a certainpattern of LDH isoenzyme elevation occurs in myocardial disease. In theabsence of hemolysis, an LDH-1/LDH-2 ratio greater than 1 (referred toas a “flipped” ratio) is usually indicative of acute myocardialinfarction (AMI). This flipped ratio is observed in about 80% of AMIpatients. The normal ratio rarely exceeds 0.80 in the absence of AMI.LDH activity begins to rise 8-12 hrs after the onset of chest pain,peaking at 24-48 hrs, and elevated levels may persist for 1 week ormore.

[0021] LDH activity and its isoenzyme pattern are also useful indicatorsof pathological conditions in the lungs, such as cell damage orinflammation. Measurement of LDH activity levels and its isoenzymepattern in pleural effusion and in bronchoalveolar lavage fluid mayprovide substantial information about lung and pulmonary endothelialcell injury.

[0022] LDH isoenzymes are also useful for evaluating other physiologicalconditions and disorders such as muscle trauma, liver damage, and avariety of malignancies. In these cases, variations of isoenzymedistribution are useful diagnostic indicators. Serum LDH levels are alsouseful for diagnosing ovarian dysgerminoma and testicular germ celltumors. Furthermore, LDH is also useful for establishing the survivalduration and rate in Hodgkin's disease and non-Hodgkin's lymphoma, andin the follow-up of ovarian dysgerminoma. Additionally, LDH is alsouseful for a variety of NAD, NADH, NADP and NADPH related studies.

[0023] For a further review of lactate dehydrogenases, see: Li, ProgClin Biol Res 1990;344:75-99; Markert et al., Cell Biochem Funct 1984July;2(3):131-4; Huijgen et al., Eur J Clin Chem Clin Biochem 1997August;35(8):569-79; Drent et al., Eur Respir J 1996August;9(8):1736-42; Vanderlinde, Ann Clin Lab Sci 1985January-February;15(1):13-31; and Wolf, Clin Lab Med 1989December;9(4):655-65.

[0024] Secreted proteins, particularly members of the lactatedehydrogenase secreted protein subfamily, are a major target for drugaction and development. Accordingly, it is valuable to the field ofpharmaceutical development to identify and characterize previouslyunknown members of this subfamily of secreted proteins. The presentinvention advances the state of the art by providing previouslyunidentified human secreted proteins that have homology to members ofthe lactate dehydrogenase secreted protein subfamily.

SUMMARY OF THE INVENTION

[0025] The present invention is based in part on the identification ofamino acid sequences of human secreted peptides and proteins that arerelated to the lactate dehydrogenase secreted protein subfamily, as wellas allelic variants and other mammalian orthologs thereof. These uniquepeptide sequences, and nucleic acid sequences that encode thesepeptides, can be used as models for the development of human therapeutictargets, aid in the identification of therapeutic proteins, and serve astargets for the development of human therapeutic agents that modulatesecreted protein activity in cells and tissues that express the secretedprotein.

DESCRIPTION OF THE FIGURE SHEETS

[0026]FIG. 1 provides the nucleotide sequence of a cDNA molecule ortranscript sequence that encodes the secreted protein of the presentinvention. (SEQ ID NO:1) In addition, structure and functionalinformation is provided, such as ATG start, stop and tissuedistribution, where available, that allows one to readily determinespecific uses of inventions based on this molecular sequence.

[0027]FIG. 2 provides the predicted amino acid sequence of the secretedprotein of the present invention. (SEQ ID NO:2) In addition structureand functional information such as protein family, function, andmodification sites is provided where available, allowing one to readilydetermine specific uses of inventions based on this molecular sequence.

[0028]FIG. 3 provides genomic sequences that span the gene encoding thesecreted protein of the present invention. (SEQ ID NO:3) In additionstructure and functional information, such as intron/exon structure,promoter location, etc., is provided where available, allowing one toreadily determine specific uses of inventions based on this molecularsequence. As indicated by the data presented in FIG. 3, the map positionwas determined to be on chromosome 15.

DETAILED DESCRIPTION OF THE INVENTION

[0029] General Description

[0030] The present invention is based on the sequencing of the humangenome. During the sequencing and assembly of the human genome, analysisof the sequence information revealed previously unidentified fragmentsof the human genome that encode peptides that share structural and/orsequence homology to protein/peptide/domains identified andcharacterized within the art as being a secreted protein or part of asecreted protein and are related to the lactate dehydrogenase secretedprotein subfamily. Utilizing these sequences, additional genomicsequences were assembled and transcript and/or cDNA sequences wereisolated and characterized. Based on this analysis, the presentinvention provides amino acid sequences of human secreted peptides andproteins that are related to the lactate dehydrogenase secreted proteinsubfamily, nucleic acid sequences in the form of transcript sequences,cDNA sequences and/or genomic sequences that encode these secretedpeptides and proteins, nucleic acid variation (allelic information),tissue distribution of expression, and information about the closest artknown protein/peptide/domain that has structural or sequence homology tothe secreted protein of the present invention.

[0031] In addition to being previously unknown, the peptides that areprovided in the present invention are selected based on their ability tobe used for the development of commercially important products andservices. Specifically, the present peptides are selected based onhomology and/or structural relatedness to known secreted proteins of thelactate dehydrogenase secreted protein subfamily and the expressionpattern observed. The art has clearly established the commercialimportance of members of this family of proteins and proteins that haveexpression patterns similar to that of the present gene. Some of themore specific features of the peptides of the present invention, and theuses thereof, are described herein, particularly in the Background ofthe Invention and in the annotation provided in the Figures, and/or areknown within the art for each of the known lactate dehydrogenase familyor subfamily of secreted proteins.

[0032] Specific Embodiments

[0033] Peptide Molecules The present invention provides nucleic acidsequences that encode protein molecules that have been identified asbeing members of the secreted protein family of proteins and are relatedto the lactate dehydrogenase secreted protein subfamily (proteinsequences are provided in FIG. 2, transcript/cDNA sequences are providedin FIG. 1 and genomic sequences are provided in FIG. 3). The peptidesequences provided in FIG. 2, as well as the obvious variants describedherein, particularly allelic variants as identified herein and using theinformation in FIG. 3, will be referred herein as the secreted peptidesof the present invention, secreted peptides, or peptides/proteins of thepresent invention.

[0034] The present invention provides isolated peptide and proteinmolecules that consist of, consist essentially of, or comprise the aminoacid sequences of the secreted peptides disclosed in the FIG. 2,(encoded by the nucleic acid molecule shown in FIG. 1, transcript/cDNAor FIG. 3, genomic sequence), as well as all obvious variants of thesepeptides that are within the art to make and use. Some of these variantsare described in detail below.

[0035] As used herein, a peptide is said to be “isolated” or “purified”when it is substantially free of cellular material or free of chemicalprecursors or other chemicals. The peptides of the present invention canbe purified to homogeneity or other degrees of purity. The level ofpurification will be based on the intended use. The critical feature isthat the preparation allows for the desired function of the peptide,even if in the presence of considerable amounts of other components (thefeatures of an isolated nucleic acid molecule is discussed below).

[0036] In some uses, “substantially free of cellular material” includespreparations of the peptide having less than about 30% (by dry weight)other proteins (i.e., contaminating protein), less than about 20% otherproteins, less than about 10% other proteins, or less than about 5%other proteins. When the peptide is recombinantly produced, it can alsobe substantially free of culture medium, i.e., culture medium representsless than about 20% of the volume of the protein preparation.

[0037] The language “substantially free of chemical precursors or otherchemicals” includes preparations of the peptide in which it is separatedfrom chemical precursors or other chemicals that are involved in itssynthesis. In one embodiment, the language “substantially free ofchemical precursors or other chemicals” includes preparations of thesecreted peptide having less than about 30% (by dry weight) chemicalprecursors or other chemicals, less than about 20% chemical precursorsor other chemicals, less than about 10% chemical precursors or otherchemicals, or less than about 5% chemical precursors or other chemicals.

[0038] The isolated secreted peptide can be purified from cells thatnaturally express it, purified from cells that have been altered toexpress it (recombinant), or synthesized using known protein synthesismethods. For example, a nucleic acid molecule encoding the secretedpeptide is cloned into an expression vector, the expression vectorintroduced into a host cell and the protein expressed in the host cell.The protein can then be isolated from the cells by an appropriatepurification scheme using standard protein purification techniques. Manyof these techniques are described in detail below.

[0039] Accordingly, the present invention provides proteins that consistof the amino acid sequences provided in FIG. 2 (SEQ ID NO:2), forexample, proteins encoded by the transcript/cDNA nucleic acid sequencesshown in FIG. 1 (SEQ ID NO:1) and the genomic sequences provided in FIG.3 (SEQ ID NO:3). The amino acid sequence of such a protein is providedin FIG. 2. A protein consists of an amino acid sequence when the aminoacid sequence is the final amino acid sequence of the protein.

[0040] The present invention further provides proteins that consistessentially of the amino acid sequences provided in FIG. 2 (SEQ IDNO:2), for example, proteins encoded by the transcript/cDNA nucleic acidsequences shown in FIG. 1 (SEQ ID NO:1) and the genomic sequencesprovided in FIG. 3 (SEQ ID NO:3). A protein consists essentially of anamino acid sequence when such an amino acid sequence is present withonly a few additional amino acid residues, for example from about 1 toabout 100 or so additional residues, typically from 1 to about 20additional residues in the final protein.

[0041] The present invention further provides proteins that comprise theamino acid sequences provided in FIG. 2 (SEQ ID NO:2), for example,proteins encoded by the transcript/cDNA nucleic acid sequences shown inFIG. 1 (SEQ ID NO:1) and the genomic sequences provided in FIG. 3 (SEQID NO:3). A protein comprises an amino acid sequence when the amino acidsequence is at least part of the final amino acid sequence of theprotein. In such a fashion, the protein can be only the peptide or haveadditional amino acid molecules, such as amino acid residues (contiguousencoded sequence) that are naturally associated with it or heterologousamino acid residues/peptide sequences. Such a protein can have a fewadditional amino acid residues or can comprise several hundred or moreadditional amino acids. The preferred classes of proteins that arecomprised of the secreted peptides of the present invention are thenaturally occurring mature proteins. A brief description of how varioustypes of these proteins can be made/isolated is provided below.

[0042] The secreted peptides of the present invention can be attached toheterologous sequences to form chimeric or fusion proteins. Suchchimeric and fusion proteins comprise a secreted peptide operativelylinked to a heterologous protein having an amino acid sequence notsubstantially homologous to the secreted peptide. “Operatively linked”indicates that the secreted peptide and the heterologous protein arefused in-frame. The heterologous protein can be fused to the N-terminusor C-terminus of the secreted peptide.

[0043] In some uses, the fusion protein does not affect the activity ofthe secreted peptide per se. For example, the fusion protein caninclude, but is not limited to, enzymatic fusion proteins, for examplebeta-galactosidase fusions, yeast two-hybrid GAL fusions, poly-Hisfusions, MYC-tagged, HI-tagged and Ig fusions. Such fusion proteins,particularly poly-His fusions, can facilitate the purification ofrecombinant secreted peptide. In certain host cells (e.g., mammalianhost cells), expression and/or secretion of a protein can be increasedby using a heterologous signal sequence.

[0044] A chimeric or fusion protein can be produced by standardrecombinant DNA techniques. For example, DNA fragments coding for thedifferent protein sequences are ligated together in-frame in accordancewith conventional techniques. In another embodiment, the fusion gene canbe synthesized by conventional techniques including automated DNAsynthesizers. Alternatively, PCR amplification of gene fragments can becarried out using anchor primers which give rise to complementaryoverhangs between two consecutive gene fragments which can subsequentlybe annealed and re-amplified to generate a chimeric gene sequence (seeAusubel et al., Current Protocols in Molecular Biology, 1992). Moreover,many expression vectors are commercially available that already encode afusion moiety (e.g., a GST protein). A secreted peptide-encoding nucleicacid can be cloned into such an expression vector such that the fusionmoiety is linked in-frame to the secreted peptide.

[0045] As mentioned above, the present invention also provides andenables obvious variants of the amino acid sequence of the proteins ofthe present invention, such as naturally occurring mature forms of thepeptide, allelic/sequence variants of the peptides, non-naturallyoccurring recombinantly derived variants of the peptides, and orthologsand paralogs of the peptides. Such variants can readily be generatedusing art-known techniques in the fields of recombinant nucleic acidtechnology and protein biochemistry. It is understood, however, thatvariants exclude any amino acid sequences disclosed prior to theinvention.

[0046] Such variants can readily be identified/made using moleculartechniques and the sequence information disclosed herein. Further, suchvariants can readily be distinguished from other peptides based onsequence and/or structural homology to the secreted peptides of thepresent invention. The degree of homology/identity present will be basedprimarily on whether the peptide is a functional variant ornon-functional variant, the amount of divergence present in the paralogfamily and the evolutionary distance between the orthologs.

[0047] To determine the percent identity of two amino acid sequences ortwo nucleic acid sequences, the sequences are aligned for optimalcomparison purposes (e.g., gaps can be introduced in one or both of afirst and a second amino acid or nucleic acid sequence for optimalalignment and non-homologous sequences can be disregarded for comparisonpurposes). In a preferred embodiment, at least 30%, 40%, 50%, 60%, 70%,80%, or 90% or more of the length of a reference sequence is aligned forcomparison purposes. The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position (asused herein amino acid or nucleic acid “identity” is equivalent to aminoacid or nucleic acid “homology”). The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which need to be introduced for optimal alignment of the twosequences.

[0048] The comparison of sequences and determination of percent identityand similarity between two sequences can be accomplished using amathematical algorithm. (Computational Molecular Biology, Lesk, A. M.,ed., Oxford University Press, New York, 1988; Biocomputing: Informaticsand Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of sequence Data, Part 1, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991). In a preferred embodiment, the percent identity betweentwo amino acid sequences is determined using the Needleman and Wunsch(J. Mol. Biol. (48):444-453 (1970)) algorithm which has beenincorporated into the GAP program in the GCG software package (availableat http://www.gcg.com), using either a Blossom 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, thepercent identity between two nucleotide sequences is determined usingthe GAP program in the GCG software package (Devereux, J., et al.,Nucleic Acids Res. 12(1):387 (1984)) (available at http://www.gcg.com),using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, thepercent identity between two amino acid or nucleotide sequences isdetermined using the algorithm of E. Myers and W. Miller (CABIOS,4:11-17 (1989)) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4.

[0049] The nucleic acid and protein sequences of the present inventioncan further be used as a “query sequence” to perform a search againstsequence databases to, for example, identify other family members orrelated sequences. Such searches can be performed using the NBLAST andXBLAST programs (version 2.0) of Altschul, et al. (J. Mol. Biol.215:403-10 (1990)). BLAST nucleotide searches can be performed with theNBLAST program, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to the nucleic acid molecules of the invention. BLAST proteinsearches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to the proteinsof the invention. To obtain gapped alignments for comparison purposes,Gapped BLAST can be utilized as described in Altschul et al. (NucleicAcids Res. 25(17):3389-3402 (1997)). When utilizing BLAST and gappedBLAST programs, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used.

[0050] Full-length pre-processed forms, as well as mature processedforms, of proteins that comprise one of the peptides of the presentinvention can readily be identified as having complete sequence identityto one of the secreted peptides of the present invention as well asbeing encoded by the same genetic locus as the secreted peptide providedherein.

[0051] Allelic variants of a secreted peptide can readily be identifiedas being a human protein having a high degree (significant) of sequencehomology/identity to at least a portion of the secreted peptide as wellas being encoded by the same genetic locus as the secreted peptideprovided herein. Genetic locus can readily be determined based on thegenomic information provided in FIG. 3, such as the genomic sequencemapped to the reference human. As indicated by the data presented inFIG. 3, the map position was determined to be on chromosome 15. As usedherein, two proteins (or a region of the proteins) have significanthomology when the amino acid sequences are typically at least about70-80%, 80-90%, and more typically at least about 90-95% or morehomologous. A significantly homologous amino acid sequence, according tothe present invention, will be encoded by a nucleic acid sequence thatwill hybridize to a secreted peptide encoding nucleic acid moleculeunder stringent conditions as more fully described below.

[0052] Paralogs of a secreted peptide can readily be identified ashaving some degree of significant sequence homology/identity to at leasta portion of the secreted peptide, as being encoded by a gene fromhumans, and as having similar activity or function. Two proteins willtypically be considered paralogs when the amino acid sequences aretypically at least about 60% or greater, and more typically at leastabout 70% or greater homology through a given region or domain. Suchparalogs will be encoded by a nucleic acid sequence that will hybridizeto a secreted peptide encoding nucleic acid molecule under moderate tostringent conditions as more fully described below.

[0053] Orthologs of a secreted peptide can readily be identified ashaving some degree of significant sequence homology/identity to at leasta portion of the secreted peptide as well as being encoded by a genefrom another organism. Preferred orthologs will be isolated frommammals, preferably primates, for the development of human therapeutictargets and agents. Such orthologs will be encoded by a nucleic acidsequence that will hybridize to a secreted peptide encoding nucleic acidmolecule under moderate to stringent conditions, as more fully describedbelow, depending on the degree of relatedness of the two organismsyielding the proteins.

[0054] Non-naturally occurring variants of the secreted peptides of thepresent invention can readily be generated using recombinant techniques.Such variants include, but are not limited to deletions, additions andsubstitutions in the amino acid sequence of the secreted peptide. Forexample, one class of substitutions are conserved amino acidsubstitution. Such substitutions are those that substitute a given aminoacid in a secreted peptide by another amino acid of likecharacteristics. Typically seen as conservative substitutions are thereplacements, one for another, among the aliphatic amino acids Ala, Val,Leu, and Ile; interchange of the hydroxyl residues Ser and Thr, exchangeof the acidic residues Asp and Glu; substitution between the amideresidues Asn and Gln; exchange of the basic residues Lys and Arg; andreplacements among the aromatic residues Phe and Tyr. Guidanceconcerning which amino acid changes are likely to be phenotypicallysilent are found in Bowie et al., Science 247:1306-1310 (1990).

[0055] Variant secreted peptides can be fully functional or can lackfunction in one or more activities, e.g. ability to bind substrate,ability to phosphorylate substrate, ability to mediate signaling, etc.Fully functional variants typically contain only conservative variationor variation in non-critical residues or in non-critical regions. FIG. 2provides the result of protein analysis and can be used to identifycritical domains/regions. Functional variants can also containsubstitution of similar amino acids that result in no change or aninsignificant change in function. Alternatively, such substitutions maypositively or negatively affect function to some degree.

[0056] Non-functional variants typically contain one or morenon-conservative amino acid substitutions, deletions, insertions,inversions, or truncation or a substitution, insertion, inversion, ordeletion in a critical residue or critical region.

[0057] Amino acids that are essential for function can be identified bymethods known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham et al., Science 244:1081-1085(1989)), particularly using the results provided in FIG. 2. The latterprocedure introduces single alanine mutations at every residue in themolecule. The resulting mutant molecules are then tested for biologicalactivity such as secreted protein activity or in assays such as an invitro proliferative activity. Sites that are critical for bindingpartner/substrate binding can also be determined by structural analysissuch as crystallization, nuclear magnetic resonance or photoaffinitylabeling (Smith et al., J. Mol. Biol. 224:899-904 (1992); de Vos et al.Science 255:306-312 (1992)).

[0058] The present invention further provides fragments of the secretedpeptides, in addition to proteins and peptides that comprise and consistof such fragments, particularly those comprising the residues identifiedin FIG. 2. The fragments to which the invention pertains, however, arenot to be construed as encompassing fragments that may be disclosedpublicly prior to the present invention.

[0059] As used herein, a fragment comprises at least 8, 10, 12, 14, 16,or more contiguous amino acid residues from a secreted peptide. Suchfragments can be chosen based on the ability to retain one or more ofthe biological activities of the secreted peptide or could be chosen forthe ability to perform a function, e.g. bind a substrate or act as animmunogen. Particularly important fragments are biologically activefragments, peptides that are, for example, about 8 or more amino acidsin length. Such fragments will typically comprise a domain or motif ofthe secreted peptide, e.g., active site or a substrate-binding domain.Further, possible fragments include, but are not limited to, domain ormotif containing fragments, soluble peptide fragments, and fragmentscontaining immunogenic structures. Predicted domains and functionalsites are readily identifiable by computer programs well known andreadily available to those of skill in the art (e.g., PROSITE analysis).The results of one such analysis are provided in FIG. 2.

[0060] Polypeptides often contain amino acids other than the 20 aminoacids commonly referred to as the 20 naturally occurring amino acids.Further, many amino acids, including the terminal amino acids, may bemodified by natural processes, such as processing and otherpost-translational modifications, or by chemical modification techniqueswell known in the art. Common modifications that occur naturally insecreted peptides are described in basic texts, detailed monographs, andthe research literature, and they are well known to those of skill inthe art (some of these features are identified in FIG. 2).

[0061] Known modifications include, but are not limited to, acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent crosslinks, formation of cystine, formation ofpyroglutamate, formylation, gamma carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination.

[0062] Such modifications are well known to those of skill in the artand have been described in great detail in the scientific literature.Several particularly common modifications, glycosylation, lipidattachment, sulfation, gamma-carboxylation of glutamic acid residues,hydroxylation and ADP-ribosylation, for instance, are described in mostbasic texts, such as Proteins—Structure and Molecular Properties, 2ndEd., T. E. Creighton, W. H. Freeman and Company, New York (1993). Manydetailed reviews are available on this subject, such as by Wold, F.,Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed.,Academic Press, New York 1-12 (1983); Seifter et al. (Meth. Enzymol.182: 626-646 (1990)) and Rattan et al. (Ann. N.Y. Acad. Sci. 663:48-62(1992)).

[0063] Accordingly, the secreted peptides of the present invention alsoencompass derivatives or analogs in which a substituted amino acidresidue is not one encoded by the genetic code, in which a substituentgroup is included, in which the mature secreted peptide is fused withanother compound, such as a compound to increase the half-life of thesecreted peptide (for example, polyethylene glycol), or in which theadditional amino acids are fused to the mature secreted peptide, such asa leader or secretory sequence or a sequence for purification of themature secreted peptide or a pro-protein sequence.

[0064] Protein/Peptide Uses

[0065] The proteins of the present invention can be used in substantialand specific assays related to the functional information provided inthe Figures; to raise antibodies or to elicit another immune response;as a reagent (including the labeled reagent) in assays designed toquantitatively determine levels of the protein (or its binding partneror ligand) in biological fluids; and as markers for tissues in which thecorresponding protein is preferentially expressed (either constitutivelyor at a particular stage of tissue differentiation or development or ina disease state). Where the protein binds or potentially binds toanother protein or ligand (such as, for example, in a secretedprotein-effector protein interaction or secreted protein-ligandinteraction), the protein can be used to identify the bindingpartner/ligand so as to develop a system to identify inhibitors of thebinding interaction. Any or all of these uses are capable of beingdeveloped into reagent grade or kit format for commercialization ascommercial products.

[0066] Methods for performing the uses listed above are well known tothose skilled in the art. References disclosing such methods include“Molecular Cloning: A Laboratory Manual”, 2d ed., Cold Spring HarborLaboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatis eds.,1989, and “Methods in Enzymology: Guide to Molecular CloningTechniques”, Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987.

[0067] The potential uses of the peptides of the present invention arebased primarily on the source of the protein as well as the class/actionof the protein. For example, secreted proteins isolated from humans andtheir human/mammalian orthologs serve as targets for identifying agentsfor use in mammalian therapeutic applications, e.g. a human drug,particularly in modulating a biological or pathological response in acell or tissue that expresses the secreted protein. A large percentageof pharmaceutical agents are being developed that modulate the activityof secreted proteins, particularly members of the lactate dehydrogenasesubfamily (see Background of the Invention). The structural andfunctional information provided in the Background and Figures providespecific and substantial uses for the molecules of the presentinvention, particularly in combination with the expression informationprovided in FIG. 1. Such uses can readily be determined using theinformation provided herein, that which is known in the art, and routineexperimentation.

[0068] The proteins of the present invention (including variants andfragments that may have been disclosed prior to the present invention)are useful for biological assays related to secreted proteins that arerelated to members of the lactate dehydrogenase subfamily. Such assaysinvolve any of the known secreted protein functions or activities orproperties useful for diagnosis and treatment of secretedprotein-related conditions that are specific for the subfamily ofsecreted proteins that the one of the present invention belongs to,particularly in cells and tissues that express the secreted protein.

[0069] The proteins of the present invention are also useful in drugscreening assays, in cell-based or cell-free systems. Cell-based systemscan be native, i.e., cells that normally express the secreted protein,as a biopsy or expanded in cell culture. In an alternate embodiment,cell-based assays involve recombinant host cells expressing the secretedprotein.

[0070] The polypeptides can be used to identify compounds that modulatesecreted protein activity of the protein in its natural state or analtered form that causes a specific disease or pathology associated withthe secreted protein. Both the secreted proteins of the presentinvention and appropriate variants and fragments can be used inhigh-throughput screens to assay candidate compounds for the ability tobind to the secreted protein. These compounds can be further screenedagainst a functional secreted protein to determine the effect of thecompound on the secreted protein activity. Further, these compounds canbe tested in animal or invertebrate systems to determineactivity/effectiveness. Compounds can be identified that activate(agonist) or inactivate (antagonist) the secreted protein to a desireddegree.

[0071] Further, the proteins of the present invention can be used toscreen a compound for the ability to stimulate or inhibit interactionbetween the secreted protein and a molecule that normally interacts withthe secreted protein, e.g. a substrate or a component of the signalpathway that the secreted protein normally interacts (for example,another secreted protein). Such assays typically include the steps ofcombining the secreted protein with a candidate compound underconditions that allow the secreted protein, or fragment, to interactwith the target molecule, and to detect the formation of a complexbetween the protein and the target or to detect the biochemicalconsequence of the interaction with the secreted protein and the target.

[0072] Candidate compounds include, for example, 1) peptides such assoluble peptides, including Ig-tailed fusion peptides and members ofrandom peptide libraries (see, e.g., Lam et al., Nature 354:82-84(1991); Houghten et al., Nature 354:84-86 (1991)) and combinatorialchemistry-derived molecular libraries made of D- and/or L-configurationamino acids; 2) phosphopeptides (e.g., members of random and partiallydegenerate, directed phosphopeptide libraries, see, e.g., Songyang etal., Cell 72:767-778 (1993)); 3) antibodies (e.g., polyclonal,monoclonal, humanized, anti-idiotypic, chimeric, and single chainantibodies as well as Fab, F(ab′)₂, Fab expression library fragments,and epitope-binding fragments of antibodies); and 4) small organic andinorganic molecules (e.g., molecules obtained from combinatorial andnatural product libraries).

[0073] One candidate compound is a soluble fragment of the receptor thatcompetes for substrate binding. Other candidate compounds include mutantsecreted proteins or appropriate fragments containing mutations thataffect secreted protein function and thus compete for substrate.Accordingly, a fragment that competes for substrate, for example with ahigher affinity, or a fragment that binds substrate but does not allowrelease, is encompassed by the invention.

[0074] Any of the biological or biochemical functions mediated by thesecreted protein can be used as an endpoint assay. These include all ofthe biochemical or biochemical/biological events described herein, inthe references cited herein, incorporated by reference for theseendpoint assay targets, and other functions known to those of ordinaryskill in the art or that can be readily identified using the informationprovided in the Figures, particularly FIG. 2. Specifically, a biologicalfunction of a cell or tissues that expresses the secreted protein can beassayed.

[0075] Binding and/or activating compounds can also be screened by usingchimeric secreted proteins in which the amino terminal extracellulardomain, or parts thereof, the entire transmembrane domain or subregions,such as any of the seven transmembrane segments or any of theintracellular or extracellular loops and the carboxy terminalintracellular domain, or parts thereof, can be replaced by heterologousdomains or subregions. For example, a substrate-binding region can beused that interacts with a different substrate then that which isrecognized by the native secreted protein. Accordingly, a different setof signal transduction components is available as an end-point assay foractivation. This allows for assays to be performed in other than thespecific host cell from which the secreted protein is derived.

[0076] The proteins of the present invention are also useful incompetition binding assays in methods designed to discover compoundsthat interact with the secreted protein (e.g. binding partners and/orligands). Thus, a compound is exposed to a secreted protein polypeptideunder conditions that allow the compound to bind or to otherwiseinteract with the polypeptide. Soluble secreted protein polypeptide isalso added to the mixture if the test compound interacts with thesoluble secreted protein polypeptide, it decreases the amount of complexformed or activity from the secreted protein target. This type of assayis particularly useful in cases in which compounds are sought thatinteract with specific regions of the secreted protein. Thus, thesoluble polypeptide that competes with the target secreted proteinregion is designed to contain peptide sequences corresponding to theregion of interest.

[0077] To perform cell free drug screening assays, it is sometimesdesirable to immobilize either the secreted protein, or fragment, or itstarget molecule to facilitate separation of complexes from uncomplexedforms of one or both of the proteins, as well as to accommodateautomation of the assay.

[0078] Techniques for immobilizing proteins on matrices can be used inthe drug screening assays. In one embodiment, a fusion protein can beprovided which adds a domain that allows the protein to be bound to amatrix. For example, glutathione-S-transferase fusion proteins can beadsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis,Mo.) or glutathione derivatized microtitre plates, which are thencombined with the cell lysates (e.g., ³⁵S-labeled) and the candidatecompound, and the mixture incubated under conditions conducive tocomplex formation (e.g., at physiological conditions for salt and pH).Following incubation, the beads are washed to remove any unbound label,and the matrix immobilized and radiolabel determined directly, or in thesupernatant after the complexes are dissociated. Alternatively, thecomplexes can be dissociated from the matrix, separated by SDS-PAGE, andthe level of secreted protein-binding protein found in the bead fractionquantitated from the gel using standard electrophoretic techniques. Forexample, either the polypeptide or its target molecule can beimmobilized utilizing conjugation of biotin and streptavidin usingtechniques well known in the art. Alternatively, antibodies reactivewith the protein but which do not interfere with binding of the proteinto its target molecule can be derivatized to the wells of the plate, andthe protein trapped in the wells by antibody conjugation. Preparationsof a secreted protein-binding protein and a candidate compound areincubated in the secreted protein-presenting wells and the amount ofcomplex trapped in the well can be quantitated. Methods for detectingsuch complexes, in addition to those described above for theGST-immobilized complexes, include immunodetection of complexes usingantibodies reactive with the secreted protein target molecule, or whichare reactive with secreted protein and compete with the target molecule,as well as enzyme-linked assays which rely on detecting an enzymaticactivity associated with the target molecule.

[0079] Agents that modulate one of the secreted proteins of the presentinvention can be identified using one or more of the above assays, aloneor in combination. It is generally preferable to use a cell-based orcell free system first and then confirm activity in an animal or othermodel system. Such model systems are well known in the art and canreadily be employed in this context.

[0080] Modulators of secreted protein activity identified according tothese drug screening assays can be used to treat a subject with adisorder mediated by the secreted protein pathway, by treating cells ortissues that express the secreted protein. These methods of treatmentinclude the steps of administering a modulator of secreted proteinactivity in a pharmaceutical composition to a subject in need of suchtreatment, the modulator being identified as described herein.

[0081] In yet another aspect of the invention, the secreted proteins canbe used as “bait proteins” in a two-hybrid assay or three-hybrid assay(see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartelet al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene8:1693-1696; and Brent WO94/10300), to identify other proteins, whichbind to or interact with the secreted protein and are involved insecreted protein activity.

[0082] The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for a secreted proteinis fused to a gene encoding the DNA binding domain of a knowntranscription factor (e.g., GAL-4). In the other construct, a DNAsequence, from a library of DNA sequences, that encodes an unidentifiedprotein (“prey” or “sample”) is fused to a gene that codes for theactivation domain of the known transcription factor. If the “bait” andthe “prey” proteins are able to interact, in vivo, forming a secretedprotein-dependent complex, the DNA-binding and activation domains of thetranscription factor are brought into close proximity. This proximityallows transcription of a reporter gene (e.g., LacZ) which is operablylinked to a transcriptional regulatory site responsive to thetranscription factor. Expression of the reporter gene can be detectedand cell colonies containing the functional transcription factor can beisolated and used to obtain the cloned gene which encodes the proteinwhich interacts with the secreted protein.

[0083] This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein inan appropriate animal model. For example, an agent identified asdescribed herein (e.g., a secreted protein-modulating agent, anantisense secreted protein nucleic acid molecule, a secretedprotein-specific antibody, or a secreted protein-binding partner) can beused in an animal or other model to determine the efficacy, toxicity, orside effects of treatment with such an agent. Alternatively, an agentidentified as described herein can be used in an animal or other modelto determine the mechanism of action of such an agent. Furthermore, thisinvention pertains to uses of novel agents identified by theabove-described screening assays for treatments as described herein.

[0084] The secreted proteins of the present invention are also useful toprovide a target for diagnosing a disease or predisposition to diseasemediated by the peptide. Accordingly, the invention provides methods fordetecting the presence, or levels of, the protein (or encoding mRNA) ina cell, tissue, or organism. The method involves contacting a biologicalsample with a compound capable of interacting with the secreted proteinsuch that the interaction-can be detected. Such an assay can be providedin a single detection format or a multi-detection format such as anantibody chip array.

[0085] One agent for detecting a protein in a sample is an antibodycapable of selectively binding to protein. A biological sample includestissues, cells and biological fluids isolated from a subject, as well astissues, cells and fluids present within a subject.

[0086] The peptides of the present invention also provide targets fordiagnosing active protein activity, disease, or predisposition todisease, in a patient having a variant peptide, particularly activitiesand conditions that are known for other members of the family ofproteins to which the present one belongs. Thus, the peptide can beisolated from a biological sample and assayed for the presence of agenetic mutation that results in aberrant peptide. This includes aminoacid substitution, deletion, insertion, rearrangement, (as the result ofaberrant splicing events), and inappropriate post-translationalmodification. Analytic methods include altered electrophoretic mobility,altered tryptic peptide digest, altered secreted protein activity incell-based or cell-free assay, alteration in substrate orantibody-binding pattern, altered isoelectric point, direct amino acidsequencing, and any other of the known assay techniques useful fordetecting mutations in a protein. Such an assay can be provided in asingle detection format or a multi-detection format such as an antibodychip array.

[0087] In vitro techniques for detection of peptide include enzymelinked immunosorbent assays (ELISAs), Western blots,immunoprecipitations and immunofluorescence using a detection reagent,such as an antibody or protein binding agent. Alternatively, the peptidecan be detected in vivo in a subject by introducing into the subject alabeled anti-peptide antibody or other types of detection agent. Forexample, the antibody can be labeled with a radioactive marker whosepresence and location in a subject can be detected by standard imagingtechniques. Particularly useful are methods that detect the allelicvariant of a peptide expressed in a subject and methods which detectfragments of a peptide in a sample.

[0088] The peptides are also useful in pharmacogenomic analysis.Pharmacogenomics deal with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. See, e.g., Eichelbaum, M. (Clin. Exp.Pharmacol. Physiol. 23(10-11):983-985 (1996)), and Linder, M. W. (Clin.Chem. 43(2):254-266 (1997)). The clinical outcomes of these variationsresult in severe toxicity of therapeutic drugs in certain individuals ortherapeutic failure of drugs in certain individuals as a result ofindividual variation in metabolism. Thus, the genotype of the individualcan determine the way a therapeutic compound acts on the body or the waythe body metabolizes the compound. Further, the activity of drugmetabolizing enzymes effects both the intensity and duration of drugaction. Thus, the pharmacogenomics of the individual permit theselection of effective compounds and effective dosages of such compoundsfor prophylactic or therapeutic treatment based on the individual'sgenotype. The discovery of genetic polymorphisms in some drugmetabolizing enzymes has explained why some patients do not obtain theexpected drug effects, show an exaggerated drug effect, or experienceserious toxicity from standard drug dosages. Polymorphisms can beexpressed in the phenotype of the extensive metabolizer and thephenotype of the poor metabolizer. Accordingly, genetic polymorphism maylead to allelic protein variants of the secreted protein in which one ormore of the secreted protein functions in one population is differentfrom those in another population. The peptides thus allow a target toascertain a genetic predisposition that can affect treatment modality.Thus, in a ligand-based treatment, polymorphism may give rise to aminoterminal extracellular domains and/or other substrate-binding regionsthat are more or less active in substrate binding, and secreted proteinactivation. Accordingly, substrate dosage would necessarily be modifiedto maximize the therapeutic effect within a given population containinga polymorphism. As an alternative to genotyping, specific polymorphicpeptides could be identified.

[0089] The peptides are also useful for treating a disordercharacterized by an absence of, inappropriate, or unwanted expression ofthe protein. Accordingly, methods for treatment include the use of thesecreted protein or fragments.

[0090] Antibodies

[0091] The invention also provides antibodies that selectively bind toone of the peptides of the present invention, a protein comprising sucha peptide, as well as variants and fragments thereof. As used herein, anantibody selectively binds a target peptide when it binds the targetpeptide and does not significantly bind to unrelated proteins. Anantibody is still considered to selectively bind a peptide even if italso binds to other proteins that are not substantially homologous withthe target peptide so long as such proteins share homology with afragment or domain of the peptide target of the antibody. In this case,it would be understood that antibody binding to the peptide is stillselective despite some degree of cross-reactivity.

[0092] As used herein, an antibody is defined in terms consistent withthat recognized within the art: they are multi-subunit proteins producedby a mammalian organism in response to an antigen challenge. Theantibodies of the present invention include polyclonal antibodies andmonoclonal antibodies, as well as fragments of such antibodies,including, but not limited to, Fab or F(ab′)₂, and Fv fragments.

[0093] Many methods are known for generating and/or identifyingantibodies to a given target peptide. Several such methods are describedby Harlow, Antibodies, Cold Spring Harbor Press, (1989).

[0094] In general, to generate antibodies, an isolated peptide is usedas an immunogen and is administered to a mammalian organism, such as arat, rabbit or mouse. The full-length protein, an antigenic peptidefragment or a fusion protein can be used. Particularly importantfragments are those covering functional domains, such as the domainsidentified in FIG. 2, and domain of sequence homology or divergenceamongst the family, such as those that can readily be identified usingprotein alignment methods and as presented in the Figures.

[0095] Antibodies are preferably prepared from regions or discretefragments of the secreted proteins. Antibodies can be prepared from anyregion of the peptide as described herein. However, preferred regionswill include those involved in function/activity and/or secretedprotein/binding partner interaction. FIG. 2 can be used to identifyparticularly important regions while sequence alignment can be used toidentify conserved and unique sequence fragments.

[0096] An antigenic fragment will typically comprise at least 8contiguous amino acid residues. The antigenic peptide can comprise,however, at least 10, 12, 14, 16 or more amino acid residues. Suchfragments can be selected on a physical property, such as fragmentscorrespond to regions that are located on the surface of the protein,e.g., hydrophilic regions or can be selected based on sequenceuniqueness (see FIG. 2).

[0097] Detection on an antibody of the present invention can befacilitated by coupling (i.e., physically linking) the antibody to adetectable substance. Examples of detectable substances include variousenzymes, prosthetic groups, fluorescent materials, luminescentmaterials, bioluminescent materials, and radioactive materials. Exanplesof suitable enzymes include horseradish peroxidase, alkalinephosphatase, β-galactosidase, or acetylcholinesterase; examples ofsuitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin,and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or³H.

[0098] Antibody Uses

[0099] The antibodies can be used to isolate one of the proteins of thepresent invention by standard techniques, such as affinitychromatography or immunoprecipitation. The antibodies can facilitate thepurification of the natural protein from cells and recombinantlyproduced protein expressed in host cells. In addition, such antibodiesare useful to detect the presence of one of the proteins of the presentinvention in cells or tissues to determine the pattern of expression ofthe protein among various tissues in an organism and over the course ofnormal development. Further, such antibodies can be used to detectprotein in situ, in vitro, or in a cell lysate or supernatant in orderto evaluate the abundance and pattern of expression. Also, suchantibodies can be used to assess abnormal tissue distribution orabnormal expression during development or progression of a biologicalcondition. Antibody detection of circulating fragments of the fulllength protein can be used to identify turnover.

[0100] Further, the antibodies can be used to assess expression indisease states such as in active stages of the disease or in anindividual with a predisposition toward disease related to the protein'sfunction. When a disorder is caused by an inappropriate tissuedistribution, developmental expression, level of expression of theprotein, or expressed/processed form, the antibody can be preparedagainst the normal protein. If a disorder is characterized by a specificmutation in the protein, antibodies specific for this mutant protein canbe used to assay for the presence of the specific mutant protein.

[0101] The antibodies can also be used to assess normal and aberrantsubcellular localization of cells in the various tissues in an organism.The diagnostic uses can be applied, not only in genetic testing, butalso in monitoring a treatment modality. Accordingly, where treatment isultimately aimed at correcting expression level or the presence ofaberrant sequence and aberrant tissue distribution or developmentalexpression, antibodies directed against the protein or relevantfragments can be used to monitor therapeutic efficacy.

[0102] Additionally, antibodies are useful in pharmacogenomic analysis.Thus, antibodies prepared against polymorphic proteins can be used toidentify individuals that require modified treatment modalities. Theantibodies are also useful as diagnostic tools as an immunologicalmarker for aberrant protein analyzed by electrophoretic mobility,isoelectric point, tryptic peptide digest, and other physical assaysknown to those in the art.

[0103] The antibodies are also useful for tissue typing. Thus, where aspecific protein has been correlated with expression in a specifictissue, antibodies that are specific for this protein can be used toidentify a tissue type.

[0104] The antibodies are also useful for inhibiting protein function,for example, blocking the binding of the secreted peptide to a bindingpartner such as a substrate. These uses can also be applied in atherapeutic context in which treatment involves inhibiting the protein'sfunction. An antibody can be used, for example, to block binding, thusmodulating (agonizing or antagonizing) the peptides activity. Antibodiescan be prepared against specific fragments containing sites required forfunction or against intact protein that is associated with a cell orcell membrane. See FIG. 2 for structural information relating to theproteins of the present invention.

[0105] The invention also encompasses kits for using antibodies todetect the presence of a protein in a biological sample. The kit cancomprise antibodies such as a labeled or labelable antibody and acompound or agent for detecting protein in a biological sample; meansfor determining the amount of protein in the sample; means for comparingthe amount of protein in the sample with a standard; and instructionsfor use. Such a kit can be supplied to detect a single protein orepitope or can be configured to detect one of a multitude of epitopes,such as in an antibody detection array. Arrays are described in detailbelow for nuleic acid arrays and similar methods have been developed forantibody arrays.

[0106] Nucleic Acid Molecules

[0107] The present invention further provides isolated nucleic acidmolecules that encode a secreted peptide or protein of the presentinvention (cDNA, transcript and genomic sequence). Such nucleic acidmolecules will consist of, consist essentially of, or comprise anucleotide sequence that encodes one of the secreted peptides of thepresent invention, an allelic variant thereof, or an ortholog or paralogthereof.

[0108] As used herein, an “isolated” nucleic acid molecule is one thatis separated from other nucleic acid present in the natural source ofthe nucleic acid. Preferably, an “isolated” nucleic acid is free ofsequences which naturally flank the nucleic acid (i.e., sequenceslocated at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA ofthe organism from which the nucleic acid is derived. However, there canbe some flanking nucleotide sequences, for example up to about 5 KB, 4KB, 3 KB, 2 KB, or 1 KB or less, particularly contiguous peptideencoding sequences and peptide encoding sequences within the same genebut separated by introns in the genomic sequence. The important point isthat the nucleic acid is isolated from remote and unimportant flankingsequences such that it can be subjected to the specific manipulationsdescribed herein such as recombinant expression, preparation of probesand primers, and other uses specific to the nucleic acid sequences.

[0109] Moreover, an “isolated” nucleic acid molecule, such as atranscript/cDNA molecule, can be substantially free of other cellularmaterial, or culture medium when produced by recombinant techniques, orchemical precursors or other chemicals when chemically synthesized.However, the nucleic acid molecule can be fused to other coding orregulatory sequences and still be considered isolated.

[0110] For example, recombinant DNA molecules contained in a vector areconsidered isolated. Further examples of isolated DNA molecules includerecombinant DNA molecules maintained in heterologous host cells orpurified (partially or substantially) DNA molecules in solution.Isolated RNA molecules include in vivo or in vitro RNA transcripts ofthe isolated DNA molecules of the present invention. Isolated nucleicacid molecules according to the present invention further include suchmolecules produced synthetically.

[0111] Accordingly, the present invention provides nucleic acidmolecules that consist of the nucleotide sequence shown in FIG. 1 or 3(SEQ ID NO:1, transcript sequence and SEQ ID NO:3, genomic sequence), orany nucleic acid molecule that encodes the protein provided in FIG. 2,SEQ ID NO:2. A nucleic acid molecule consists of a nucleotide sequencewhen the nucleotide sequence is the complete nucleotide sequence of thenucleic acid molecule.

[0112] The present invention further provides nucleic acid moleculesthat consist essentially of the nucleotide sequence shown in FIG. 1 or 3(SEQ ID NO:1, transcript sequence and SEQ ID NO:3, genomic sequence), orany nucleic acid molecule that encodes the protein provided in FIG. 2,SEQ ID NO:2. A nucleic acid molecule consists essentially of anucleotide sequence when such a nucleotide sequence is present with onlya few additional nucleic acid residues in the final nucleic acidmolecule.

[0113] The present invention further provides nucleic acid moleculesthat comprise the nucleotide sequences shown in FIG. 1 or 3 (SEQ IDNO:1, transcript sequence and SEQ ID NO:3, genomic sequence), or anynucleic acid molecule that ericodes the protein provided in FIG. 2, SEQID NO:2. A nucleic acid molecule comprises a nucleotide sequence whenthe nucleotide sequence is at least part of the final nucleotidesequence of the nucleic acid molecule. In such a fashion, the nucleicacid molecule can be only the nucleotide sequence or have additionalnucleic acid residues, such as nucleic acid residues that are naturallyassociated with it or heterologous nucleotide sequences. Such a nucleicacid molecule can have a few additional nucleotides or can comprisesseveral hundred or more additional nucleotides. A brief description ofhow various types of these nucleic acid molecules can be readilymade/isolated is provided below.

[0114] In FIGS. 1 and 3, both coding and non-coding sequences areprovided. Because of the source of the present invention, humans genomicsequence (FIG. 3) and cDNA/transcript sequences (FIG. 1), the nucleicacid molecules in the Figures will contain genomic intronic sequences,5′ and 3′ non-coding sequences, gene regulatory regions and non-codingintergenic sequences. In general such sequence features are either notedin FIGS. 1 and 3 or can readily be identified using computational toolsknown in the art. As discussed below, some of the non-coding regions,particularly gene regulatory elements such as promoters, are useful fora variety of purposes, e.g. control of heterologous gene expression,target for identifying gene activity modulating compounds, and areparticularly claimed as fragments of the genomic sequence providedherein.

[0115] The isolated nucleic acid molecules can encode the mature proteinplus additional amino or carboxyl-terminal amino acids, or amino acidsinterior to the mature peptide (when the mature form has more than onepeptide chain, for instance). Such sequences may play a role inprocessing of a protein from precursor to a mature form, facilitateprotein trafficking, prolong or shorten protein half-life or facilitatemanipulation of a protein for assay or production, among other things.As generally is the case in situ, the additional amino acids may beprocessed away from the mature protein by cellular enzymes.

[0116] As mentioned above, the isolated nucleic acid molecules include,but are not limited to, the sequence encoding the secreted peptidealone, the sequence encoding the mature peptide and additional codingsequences, such as a leader or secretory sequence (e.g., a pre-pro orpro-protein sequence), the sequence encoding the mature peptide, with orwithout the additional coding sequences, plus additional non-codingsequences, for example introns and non-coding 5′ and 3′ sequences suchas transcribed but non-translated sequences that play a role intranscription, mRNA processing (including splicing and polyadenylationsignals), ribosome binding and stability of mRNA. In addition, thenucleic acid molecule may be fused to a marker sequence encoding, forexample, a peptide that facilitates purification.

[0117] Isolated nucleic acid molecules can be in the form of RNA, suchas mRNA, or in the form DNA, including cDNA and genomic DNA obtained bycloning or produced by chemical synthetic techniques or by a combinationthereof. The nucleic acid, especially DNA, can be double-stranded orsingle-stranded. Single-stranded nucleic acid can be the coding strand(sense strand) or the non-coding strand (anti-sense strand).

[0118] The invention further provides nucleic acid molecules that encodefragments of the peptides of the present invention as well as nucleicacid molecules that encode obvious variants of the secreted proteins ofthe present invention that are described above. Such nucleic acidmolecules may be naturally occurring, such as allelic variants (samelocus), paralogs (different locus), and orthologs (different organism),or may be constructed by recombinant DNA methods or by chemicalsynthesis. Such non-naturally occurring variants may be made bymutagenesis techniques, including those applied to nucleic acidmolecules, cells, or organisms. Accordingly, as discussed above, thevariants can contain nucleotide substitutions, deletions, inversions andinsertions. Variation can occur in either or both the coding andnon-coding regions. The variations can produce both conservative andnon-conservative amino acid substitutions.

[0119] The present invention further provides non-coding fragments ofthe nucleic acid molecules provided in FIGS. 1 and 3. Preferrednon-coding fragments include, but are not limited to, promotersequences, enhancer sequences, gene modulating sequences and genetermination sequences. Such fragments are useful in controllingheterologous gene expression and in developing screens to identifygene-modulating agents. A promoter can readily be identified as being 5′to the ATG start site in the genomic sequence provided in FIG. 3.

[0120] A fragment comprises a contiguous nucleotide sequence greaterthan 12 or more nucleotides. Further, a fragment could at least 30, 40,50, 100, 250 or 500 nucleotides in length. The length of the fragmentwill be based on its intended use. For example, the fragment can encodeepitope bearing regions of the peptide, or can be useful as DNA probesand primers. Such fragments can be isolated using the known nucleotidesequence to synthesize an oligonucleotide probe. A labeled probe canthen be used to screen a cDNA library, genomic DNA library, or mRNA toisolate nucleic acid corresponding to the coding region. Further,primers can be used in PCR reactions to clone specific regions of gene.

[0121] A probe/primer typically comprises substantially a purifiedoligonucleotide or oligonucleotide pair. The oligonucleotide typicallycomprises a region of nucleotide sequence that hybridizes understringent conditions to at least about 12, 20, 25, 40, 50 or moreconsecutive nucleotides.

[0122] Orthologs, homologs, and allelic variants can be identified usingmethods well known in the art. As described in the Peptide Section,these variants comprise a nucleotide sequence encoding a peptide that istypically 60-70%, 70-80%, 80-90%, and more typically at least about90-95% or more homologous to the nucleotide sequence shown in the Figuresheets or a fragment of this sequence. Such nucleic acid molecules canreadily be identified as being able to hybridize under moderate tostringent conditions, to the nucleotide sequence shown in the Figuresheets or a fragment of the sequence. Allelic variants can readily bedetermined by genetic locus of the encoding gene.

[0123] As used herein, the term “hybridizes under stringent conditions”is intended to describe conditions for hybridization and washing underwhich nucleotide sequences encoding a peptide at least 60-70% homologousto each other typically remain hybridized to each other. The conditionscan be such that sequences at least about 60%, at least about 70%, or atleast about 80% or more homologous to each other typically remainhybridized to each other. Such stringent conditions are known to thoseskilled in the art and can be found in Current Protocols in MolecularBiology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. One example ofstringent hybridization conditions are hybridization in 6× sodiumchloride/sodium citrate (SSC) at about 45 C, followed by one or morewashes in 0.2×SSC, 0.1% SDS at 50-65 C. Examples of moderate to lowstringency hybridization conditions are well known in the art.

[0124] Nucleic Acid Molecule Uses

[0125] The nucleic acid molecules of the present invention are usefulfor probes, primers, chemical intermediates, and in biological assays.The nucleic acid molecules are useful as a hybridization probe formessenger RNA, transcript/cDNA and genomic DNA to isolate full-lengthcDNA and genomic clones encoding the peptide described in FIG. 2 and toisolate cDNA and genomic clones that correspond to variants (alleles,orthologs, etc.) producing the same or related peptides shown in FIG. 2.

[0126] The probe can correspond to any sequence along the entire lengthof the nucleic acid molecules provided in the Figures. Accordingly, itcould be derived from 5′ noncoding regions, the coding region, and 3′noncoding regions. However, as discussed, fragments are not to beconstrued as encompassing fragments disclosed prior to the presentinvention.

[0127] The nucleic acid molecules are also useful as primers for PCR toamplify any given region of a nucleic acid molecule and are useful tosynthesize antisense molecules of desired length and sequence.

[0128] The nucleic acid molecules are also useful for constructingrecombinant vectors. Such vectors include expression vectors thatexpress a portion of, or all of, the peptide sequences. Vectors alsoinclude insertion vectors, used to integrate into another nucleic acidmolecule sequence, such as into the cellular genome, to alter in situexpression of a gene and/or gene product. For example, an endogenouscoding sequence can be replaced via homologous recombination with all orpart of the coding region containing one or more specifically introducedmutations.

[0129] The nucleic acid molecules are also useful for expressingantigenic portions of the proteins.

[0130] The nucleic acid molecules are also useful as probes fordetermining the chromosomal positions of the nucleic acid molecules bymeans of in situ hybridization methods.

[0131] The nucleic acid molecules are also useful in making vectorscontaining the gene regulatory regions of the nucleic acid molecules ofthe present invention.

[0132] The nucleic acid molecules are also useful for designingribozymes corresponding to all, or a part, of the mRNA produced from thenucleic acid molecules described herein.

[0133] The nucleic acid molecules are also useful for making vectorsthat express part, or all, of the peptides.

[0134] The nucleic acid molecules are also useful for constructing hostcells expressing a part, or all, of the nucleic acid molecules andpeptides.

[0135] The nucleic acid molecules are also useful for constructingtransgenic animals expressing all, or a part, of the nucleic acidmolecules and peptides.

[0136] The nucleic acid molecules are also useful as hybridizationprobes for determining the presence, level, form and distribution ofnucleic acid expression. Accordingly, the probes can be used to detectthe presence of, or to determine levels of, a specific nucleic acidmolecule in cells, tissues, and in organisms. The nucleic acid whoselevel is determined can be DNA or RNA. Accordingly, probes correspondingto the peptides described herein can be used to assess expression and/orgene copy number in a given cell, tissue, or organism. These uses arerelevant for diagnosis of disorders involving an increase or decrease insecreted protein expression relative to normal results.

[0137] In vitro techniques for detection of mRNA include Northernhybridizations and in situ hybridizations. In vitro techniques fordetecting DNA include Southern hybridizations and in situ hybridization.

[0138] Probes can be used as a part of a diagnostic test kit foridentifying cells or tissues that express a secreted protein, such as bymeasuring a level of a secreted protein-encoding nucleic acid in asample of cells from a subject e.g., mRNA or genomic DNA, or determiningif a secreted protein gene has been mutated.

[0139] Nucleic acid expression assays are useful for drug screening toidentify compounds that modulate secreted protein nucleic acidexpression.

[0140] The invention thus provides a method for identifying a compoundthat can be used to treat a disorder associated with nucleic acidexpression of the secreted protein gene, particularly biological andpathological processes that are mediated by the secreted protein incells and tissues that express it. The method typically includesassaying the ability of the compound to modulate the expression of thesecreted protein nucleic acid and thus identifying a compound that canbe used to treat a disorder characterized by undesired secreted proteinnucleic acid expression. The assays can be performed in cell-based andcell-free systems. Cell-based assays include cells naturally expressingthe secreted protein nucleic acid or recombinant cells geneticallyengineered to express specific nucleic acid sequences.

[0141] Thus, modulators of secreted protein gene expression can beidentified in a method wherein a cell is contacted with a candidatecompound and the expression of mRNA determined. The level of expressionof secreted protein mRNA in the presence of the candidate compound iscompared to the level of expression of secreted protein mRNA in theabsence of the candidate compound. The candidate compound can then beidentified as a modulator of nucleic acid expression based on thiscomparison and be used, for example to treat a disorder characterized byaberrant nucleic acid expression. When expression of mRNA isstatistically significantly greater in the presence of the candidatecompound than in its absence, the candidate compound is identified as astimulator of nucleic acid expression. When nucleic acid expression isstatistically significantly less in the presence of the candidatecompound than in its absence, the candidate compound is identified as aninhibitor of nucleic acid expression.

[0142] The invention further provides methods of treatment, with thenucleic acid as a target, using a compound identified through drugscreening as a gene modulator to modulate secreted protein nucleic acidexpression in cells and tissues that express the secreted protein.Modulation includes both up-regulation (i.e. activation or agonization)or down-regulation (suppression or antagonization) or nucleic acidexpression.

[0143] Alternatively, a modulator for secreted protein nucleic acidexpression can be a small molecule or drug identified using thescreening assays described herein as long as the drug or small moleculeinhibits the secreted protein nucleic acid expression in the cells andtissues that express the protein.

[0144] The nucleic acid molecules are also useful for monitoring theeffectiveness of modulating compounds on the expression or activity ofthe secreted protein gene in clinical trials or in a treatment regimen.Thus, the gene expression pattern can serve as a barometer for thecontinuing effectiveness of treatment with the compound, particularlywith compounds to which a patient can develop resistance. The geneexpression pattern can also serve as a marker indicative of aphysiological response of the affected cells to the compound.Accordingly, such monitoring would allow either increased administrationof the compound or the administration of alternative compounds to whichthe patient has not become resistant. Similarly, if the level of nucleicacid expression falls below a desirable level, administration of thecompound could be commensurately decreased.

[0145] The nucleic acid molecules are also useful in diagnostic assaysfor qualitative changes in secreted protein nucleic acid expression, andparticularly in qualitative changes that lead to pathology. The nucleicacid molecules can be used to detect mutations in secreted protein genesand gene expression products such as mRNA. The nucleic acid moleculescan be used as hybridization probes to detect naturally occurringgenetic mutations in the secreted protein gene and thereby to determinewhether a subject with the mutation is at risk for a disorder caused bythe mutation. Mutations include deletion, addition, or substitution ofone or more nucleotides in the gene, chromosomal rearrangement, such asinversion or transposition, modification of genomic DNA, such asaberrant methylation patterns or changes in gene copy number, such asamplification. Detection of a mutated form of the secreted protein geneassociated with a dysfunction provides a diagnostic tool for an activedisease or susceptibility to disease when the disease results fromoverexpression, underexpression, or altered expression of a secretedprotein.

[0146] Individuals carrying mutations in the secreted protein gene-canbe detected at the nucleic acid level by a variety of techniques.Genomic DNA can be analyzed directly or can be amplified by using PCRprior to analysis. RNA or cDNA can be used in the same way. In someuses, detection of the mutation involves the use of a probe/primer in apolymerase chain reaction (PCR) (see, e.g. U.S. Pat. Nos. 4,683,195 and4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in aligation chain reaction (LCR) (see, e.g., Landegran et al., Science241:1077-1080 (1988); and Nakazawa et al., PNAS 91:360-364 (1994)), thelatter of which can be particularly useful for detecting point mutationsin the gene (see Abravaya et al., Nucleic Acids Res. 23:675-682 (1995)).This method can include the steps of collecting a sample of cells from apatient, isolating nucleic acid (e.g., genomic, mRNA or both) from thecells of the sample, contacting the nucleic acid sample with one or moreprimers which specifically hybridize to a gene under conditions suchthat hybridization and amplification of the gene (if present) occurs,and detecting the presence or absence of an amplification product, ordetecting the size of the amplification product and comparing the lengthto a control sample. Deletions and insertions can be detected by achange in size of the amplified product compared to the normal genotype.Point mutations can be identified by hybridizing amplified DNA to normalRNA or antisense DNA sequences.

[0147] Alternatively, mutations in a secreted protein gene can bedirectly identified, for example, by alterations in restriction enzymedigestion patterns determined by gel electrophoresis.

[0148] Further, sequence-specific ribozymes (U.S. Pat. No. 5,498,531)can be used to score for the presence of specific mutations bydevelopment or loss of a ribozyme cleavage site. Perfectly matchedsequences can be distinguished from mismatched sequences by nucleasecleavage digestion assays or by differences in melting temperature.

[0149] Sequence changes at specific locations can also be assessed bynuclease protection assays such as RNase and S1 protection or thechemical cleavage method. Furthermore, sequence differences between amutant secreted protein gene and a wild-type gene can be determined bydirect DNA sequencing. A variety of automated sequencing procedures canbe utilized when performing the diagnostic assays (Naeve, C. W., (1995)Biotechniques 19:448), including sequencing by mass spectrometry (see,e.g., PCT International Publication No. WO 94/16101; Cohen et al., Adv.Chromatogr. 36:127-162 (1996); and Griffin et al., Appl. Biochem.Biotechnol. 38:147-159 (1993)).

[0150] Other methods for detecting mutations in the gene include methodsin which protection from cleavage agents is used to detect mismatchedbases in RNA/RNA or RNA/DNA duplexes (Myers et al., Science 230:1242(1985)); Cotton et al., PNAS 85:4397 (1988); Saleeba et al., Meth.Enzymol. 217:286-295 (1992)), electrophoretic mobility of mutant andwild type nucleic acid is compared (Orita et al., PNAS 86:2766 (1989);Cotton et al., Mutat. Res. 285:125-144 (1993); and Hayashi et al.,Genet. Anal. Tech. Appl. 9:73-79 (1992)), and movement of mutant orwild-type fragments in polyacrylamide gels containing a gradient ofdenaturant is assayed using denaturing gradient gel electrophoresis(Myers et al., Nature 313:495 (1985)). Examples of other techniques fordetecting point mutations include selective oligonucleotidehybridization, selective amplification, and selective primer extension.

[0151] The nucleic acid molecules are also useful for testing anindividual for a genotype that while not necessarily causing thedisease, nevertheless affects the treatment modality. Thus, the nucleicacid molecules can be used to study the relationship between anindividual's genotype and the individual's response to a compound usedfor treatment (pharmacogenomic relationship). Accordingly, the nucleicacid molecules described herein can be used to assess the mutationcontent of the secreted protein gene in an individual in order to selectan appropriate compound or dosage regimen for treatment.

[0152] Thus nucleic acid molecules displaying genetic variations thataffect treatment provide a diagnostic target that can be used to tailortreatment in an individual. Accordingly, the production of recombinantcells and animals containing these polymorphisms allow effectiveclinical design of treatment compounds and dosage regimens.

[0153] The nucleic acid molecules are thus useful as antisenseconstructs to control secreted protein gene expression in cells,tissues, and organisms. A DNA antisense nucleic acid molecule isdesigned to be complementary to a region of the gene involved intranscription, preventing transcription and hence production of secretedprotein. An antisense RNA or DNA nucleic acid molecule would hybridizeto the mRNA and thus block translation of mRNA into secreted protein.

[0154] Alternatively, a class of antisense molecules can be used toinactivate mRNA in order to decrease expression of secreted proteinnucleic acid. Accordingly, these molecules can treat a disordercharacterized by abnormal or undesired secreted protein nucleic acidexpression. This technique involves cleavage by means of ribozymescontaining nucleotide sequences complementary to one or more regions inthe mRNA that attenuate the ability of the mRNA to be translated.Possible regions include coding regions and particularly coding regionscorresponding to the catalytic and other functional activities of thesecreted protein, such as substrate binding.

[0155] The nucleic acid molecules also provide vectors for gene therapyin patients containing cells that are aberrant in secreted protein geneexpression. Thus, recombinant cells, which include the patient's cellsthat have been engineered ex vivo and returned to the patient, areintroduced into an individual where the cells produce the desiredsecreted protein to treat the individual.

[0156] The invention also encompasses kits for detecting the presence ofa secreted protein nucleic acid in a biological sample. For example, thekit can comprise reagents such as a labeled or labelable nucleic acid oragent capable of detecting secreted protein nucleic acid in a biologicalsample; means for determining the amount of secreted protein nucleicacid in the sample; and means for comparing the amount of secretedprotein nucleic acid in the sample with a standard. The compound oragent can be packaged in a suitable container. The kit can furthercomprise instructions for using the kit to detect secreted protein mRNAor DNA.

[0157] Nucleic Acid Arrays

[0158] The present invention further provides nucleic acid detectionkits, such as arrays or microarrays of nucleic acid molecules that arebased on the sequence information provided in FIGS. 1 and 3 (SEQ IDNOS:1 and 3).

[0159] As used herein “Arrays” or “Microarrays” refers to an array ofdistinct polynucleotides or oligonucleotides synthesized on a substrate,such as paper, nylon or other type of membrane, filter, chip, glassslide, or any other suitable solid support. In one embodiment, themicroarray is prepared and used according to the methods described inU.S. Pat. No. 5,837,832, Chee et al., PCT application WO95/11995 (Cheeet al.), Lockhart, D. J. et al. (1996; Nat. Biotech. 14: 1675-1680) andSchena, M. et al. (1996; Proc. Natl. Acad. Sci. 93: 10614-10619), all ofwhich are incorporated herein in their entirety by reference. In otherembodiments, such arrays are produced by the methods described by Brownet al., U.S. Pat. No. 5,807,522.

[0160] The microarray or detection kit is preferably composed of a largenumber of unique, single-stranded nucleic acid sequences, usually eithersynthetic antisense oligonucleotides or fragments of cDNAs, fixed to asolid support. The oligonucleotides are preferably about 6-60nucleotides in length, more preferably 15-30 nucleotides in length, andmost preferably about 20-25 nucleotides in length. For a certain type ofmicroarray or detection kit, it may be preferable to useoligonucleotides that are only 7-20 nucleotides in length. Themicroarray or detection kit may contain oligonucleotides that cover theknown 5′, or 3′, sequence, sequential oligonucleotides which cover thefull length sequence; or unique oligonucleotides selected fromparticular areas along the length of the sequence. Polynucleotides usedin the microarray or detection kit may be oligonucleotides that arespecific to a gene or genes of interest.

[0161] In order to produce oligonucleotides to a known sequence for amicroarray or detection kit, the gene(s) of interest (or an ORFidentified from the contigs of the present invention) is typicallyexamined using a computer algorithm which starts at the 5′ or at the 3′end of the nucleotide sequence. Typical algorithms will then identifyoligomers of defined length that are unique to the gene, have a GCcontent within a range suitable for hybridization, and lack predictedsecondary structure that may interfere with hybridization. In certainsituations it may be appropriate to use pairs of oligonucleotides on amicroarray or detection kit. The “pairs” will be identical, except forone nucleotide that preferably is located in the center of the sequence.The second oligonucleotide in the pair (mismatched by one) serves as acontrol. The number of oligonucleotide pairs may range from two to onemillion. The oligomers are synthesized at designated areas on asubstrate using a light-directed chemical process. The substrate may bepaper, nylon or other type of membrane, filter, chip, glass slide or anyother suitable solid support.

[0162] In another aspect, an oligonucleotide may be synthesized on thesurface of the substrate by using a chemical coupling procedure and anink jet application apparatus, as described in PCT applicationWO95/251116 (Baldeschweiler et al.) which is incorporated herein in itsentirety by reference. In another aspect, a “gridded” array analogous toa dot (or slot) blot may be used to arrange and link cDNA fragments oroligonucleotides to the surface of a substrate using a vacuum system,thermal, UV, mechanical or chemical bonding procedures. An array, suchas those described above, may be produced by hand or by using availabledevices (slot blot or dot blot apparatus), materials (any suitable solidsupport), and machines (including robotic instruments), and may contain8, 24, 96, 384, 1536, 6144 or more oligonucleotides, or any other numberbetween two and one million which lends itself to the efficient use ofcommercially available instrumentation.

[0163] In order to conduct sample analysis using a microarray ordetection kit, the RNA or DNA from a biological sample is made intohybridization probes. The mRNA is isolated, and cDNA is produced andused as a template to make antisense RNA (aRNA). The aRNA is amplifiedin the presence of fluorescent nucleotides, and labeled probes areincubated with the microarray or detection kit so that the probesequences hybridize to complementary oligonucleotides of the microarrayor detection kit. Incubation conditions are adjusted so thathybridization occurs with precise complementary matches or with variousdegrees of less complementarity. After removal of nonhybridized probes,a scanner is used to determine the levels and patterns of fluorescence.The scanned images are examined to determine degree of complementarityand the relative abundance of each oligonucleotide sequence on themicroarray or detection kit. The biological samples may be obtained fromany bodily fluids (such as blood, urine, saliva, phlegm, gastric juices,etc.), cultured cells, biopsies, or other tissue preparations. Adetection system may be used to measure the absence, presence, andamount of hybridization for all of the distinct sequencessimultaneously. This data may be used for large-scale correlationstudies on the sequences, expression patterns, mutations, variants, orpolymorphisms among samples.

[0164] Using such arrays, the present invention provides methods toidentify the expression of the secreted proteins/peptides of the presentinvention. In detail, such methods comprise incubating a test samplewith one or more nucleic acid molecules and assaying for binding of thenucleic acid molecule with components within the test sample. Suchassays will typically involve arrays comprising many genes, at least oneof which is a gene of the present invention and or alleles of thesecreted protein gene of the present invention.

[0165] Conditions for incubating a nucleic acid molecule with a testsample vary. Incubation conditions depend on the format employed in theassay, the detection methods employed, and the type and nature of thenucleic acid molecule used in the assay. One skilled in the art willrecognize that any one of the commonly available hybridization,amplification or array assay formats can readily be adapted to employthe novel fragments of the Human genome disclosed herein. Examples ofsuch assays can be found in Chard, T, An Introduction toRadioimmunoassay and Related Techniques, Elsevier Science Publishers,Amsterdam, The Netherlands (1986); Bullock, G. R. et al., Techniques inImmunocytochemistry, Academic Press, Orlando, Fla. Vol. 1 (1982), Vol. 2(1983), Vol. 3 (1985); Tijssen, P., Practice and Theory of EnzymeImmunoassays: Laboratory Techniques in Biochemistry and MolecularBiology, Elsevier Science Publishers, Amsterdam, The Netherlands (1985).

[0166] The test samples of the present invention include cells, proteinor membrane extracts of cells. The test sample used in theabove-described method will vary based on the assay format, nature ofthe detection method and the tissues, cells or extracts used as thesample to be assayed. Methods for preparing nucleic acid extracts or ofcells are well known in the art and can be readily be adapted in orderto obtain a sample that is compatible with the system utilized.

[0167] In another embodiment of the present invention, kits are providedwhich contain the necessary reagents to carry out the assays of thepresent invention.

[0168] Specifically, the invention provides a compartmentalized kit toreceive, in close confinement, one or more containers which comprises:(a) a first container comprising one of the nucleic acid molecules thatcan bind to a fragment of the Human genome disclosed herein; and (b) oneor more other containers comprising one or more of the following: washreagents, reagents capable of detecting presence of a bound nucleicacid.

[0169] In detail, a compartmentalized kit includes any kit in whichreagents are contained in separate containers. Such containers includesmall glass containers, plastic containers, strips of plastic, glass orpaper, or arraying material such as silica. Such containers allows oneto efficiently transfer reagents from one compartment to anothercompartment such that the samples and reagents are notcross-contaminated, and the agents or solutions of each container can beadded in a quantitative fashion from one compartment to another. Suchcontainers will include a container which will accept the test sample, acontainer which contains the nucleic acid probe, containers whichcontain wash reagents (such as phosphate buffered saline, Tris-buffers,etc.), and containers which contain the reagents used to detect thebound probe. One skilled in the art will readily recognize that thepreviously unidentified secreted protein gene of the present inventioncan be routinely identified using the sequence information disclosedherein can be readily incorporated into one of the established kitformats which are well known in the art, particularly expression arrays.

[0170] Vectors/Host Cells

[0171] The invention also provides vectors containing the nucleic acidmolecules described herein. The term “vector” refers to a vehicle,preferably a nucleic acid molecule, which can transport the nucleic acidmolecules. When the vector is a nucleic acid molecule, the nucleic acidmolecules are covalently linked to the vector nucleic acid. With thisaspect of the invention, the vector includes a plasmid, single or doublestranded phage, a single or double stranded RNA or DNA viral vector, orartificial chromosome, such as a BAC, PAC, YAC, OR MAC.

[0172] A vector can be maintained in the host cell as anextrachromosomal element where it replicates and produces additionalcopies of the nucleic acid molecules. Alternatively, the vector mayintegrate into the host cell genome and produce additional copies of thenucleic acid molecules when the host cell replicates.

[0173] The invention provides vectors for the maintenance (cloningvectors) or vectors for expression (expression vectors) of the nucleicacid molecules. The vectors can function in prokaryotic or eukaryoticcells or in both (shuttle vectors).

[0174] Expression vectors contain cis-acting regulatory regions that areoperably linked in the vector to the nucleic acid molecules such thattranscription of the nucleic acid molecules is allowed in a host cell.The nucleic acid molecules can be introduced into the host cell with aseparate nucleic acid molecule capable of affecting transcription. Thus,the second nucleic acid molecule may provide a trans-acting factorinteracting with the cis-regulatory control region to allowtranscription of the nucleic acid molecules from the vector.Alternatively, a trans-acting factor may be supplied by the host cell.Finally, a trans-acting factor can be produced from the vector itself.It is understood, however, that in some embodiments, transcriptionand/or translation of the nucleic acid molecules can occur in acell-free system.

[0175] The regulatory sequence to which the nucleic acid moleculesdescribed herein can be operably linked include promoters for directingmRNA transcription. These include, but are not limited to, the leftpromoter from bacteriophage λ, the lac, TRP, and TAC promoters from E.Coli, the early and late promoters from SV40, the CMV immediate earlypromoter, the adenovirus early and late promoters, and retroviruslong-terminal repeats.

[0176] In addition to control regions that promote transcription,expression vectors may also include regions that modulate transcription,such as repressor binding sites and enhancers. Examples include the SV40enhancer, the cytomegalovirus immediate early enhancer, polyomaenhancer, adenovirus enhancers, and retrovirus LTR enhancers.

[0177] In addition to containing sites for transcription initiation andcontrol, expression vectors can also contain sequences necessary fortranscription termination and, in the transcribed region a ribosomebinding site for translation. Other regulatory control elements forexpression include initiation and termination codons as well aspolyadenylation signals. The person of ordinary skill in the art wouldbe aware of the numerous regulatory sequences that are useful inexpression vectors. Such regulatory sequences are described, forexample, in Sambrook et al, Molecular Cloning: A Laboratory Manual. 2nd.ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,(1989).

[0178] A variety of expression vectors can be used to express a nucleicacid molecule. Such vectors include chromosomal, episomal, andvirus-derived vectors, for example vectors derived from bacterialplasmids, from bacteriophage, from yeast episomes, from yeastchromosomal elements, including yeast artificial chromosomes, fromviruses such as baculoviruses, papovaviruses such as SV40, Vacciniaviruses, adenoviruses, poxviruses, pseudorabies viruses, andretroviruses. Vectors may also be derived from combinations of thesesources such as those derived from plasmid and bacteriophage geneticelements, e.g. cosmids and phagemids. Appropriate cloning and expressionvectors for prokaryotic and eukaryotic hosts are described in Sambrooket al., Molecular Cloning: A Laboratory Manual. 2nd. ed., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., (1989).

[0179] The regulatory sequence may provide constitutive expression inone or more host cells (i.e. tissue specific) or may provide forinducible expression in one or more cell types such as by temperature,nutrient additive, or exogenous factor such as a hormone or otherligand. A variety of vectors providing for constitutive and inducibleexpression in prokaryotic and eukaryotic hosts are well known to thoseof ordinary skill in the art.

[0180] The nucleic acid molecules can be inserted into the vectornucleic acid by well-known methodology. Generally, the DNA sequence thatwill ultimately be expressed is joined to an expression vector bycleaving the DNA sequence and the expression vector with one or morerestriction enzymes and then ligating the fragments together. Proceduresfor restriction enzyme digestion and ligation are well known to those ofordinary skill in the art.

[0181] The vector containing the appropriate nucleic acid molecule canbe introduced into an appropriate host cell for propagation orexpression using well-known techniques. Bacterial cells include, but arenot limited to, E. coli, Streptomyces, and Salmonella typhimurium.Eukaryotic cells include, but are not limited to, yeast, insect cellssuch as Drosophila, animal cells such as COS and CHO cells, and plantcells.

[0182] As described herein, it may be desirable to express the peptideas a fusion protein. Accordingly, the invention provides fusion vectorsthat allow for the production of the peptides. Fusion vectors canincrease the expression of a recombinant protein, increase thesolubility of the recombinant protein, and aid in the purification ofthe protein by acting for example as a ligand for affinity purification.A proteolytic cleavage site may be introduced at the junction of thefusion moiety so that the desired peptide can ultimately be separatedfrom the fusion moiety. Proteolytic enzymes include, but are not limitedto, factor Xa, thrombin, and enterokinase. Typical fusion expressionvectors include pGEX (Smith et al., Gene 67:31-40 (1988)), pMAL (NewEngland Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.)which fuse glutathione S-transferase (GST), maltose E binding protein,or protein A, respectively, to the target recombinant protein. Examplesof suitable inducible non-fusion E. coli expression vectors include pTrc(Amann et al., Gene 69:301-315 (1988)) and pET 11d (Studier et al., GeneExpression Technology: Methods in Enzymology 185:60-89 (1990)).

[0183] Recombinant protein expression can be maximized in host bacteriaby providing a genetic background wherein the host cell has an impairedcapacity to proteolytically cleave the recombinant protein. (Gottesman,S., Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990)119-128). Alternatively, the sequence ofthe nucleic acid molecule of interest can be altered to providepreferential codon usage for a specific host cell, for example E. coli.(Wada et al., Nucleic Acids Res. 20:2111-2118 (1992)).

[0184] The nucleic acid molecules can also be expressed by expressionvectors that are operative in yeast. Examples of vectors for expressionin yeast e.g., S. cerevisiae include pYepSec1 (Baldari, et al., EMBO J.6:229-234 (1987)), pMFa (Kujan et al., Cell 30:933-943(1982)), pJRY88(Schultz et al., Gene 54:113-123 (1987)), and pYES2 (InvitrogenCorporation, San Diego, Calif.).

[0185] The nucleic acid molecules can also be expressed in insect cellsusing, for example, baculovirus expression vectors. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., Sf9 cells) include the pAc series (Smith et al., Mol. Cell Biol.3:2156-2165 (1983)) and the pVL series (Lucklow et al., Virology170:31-39 (1989)).

[0186] In certain embodiments of the invention, the nucleic acidmolecules described herein are expressed in mammalian cells usingmammalian expression vectors. Examples of mammalian expression vectorsinclude pCDM8 (Seed, B. Nature 329:840(1987)) and pMT2PC (Kaufman etal., EMBO J. 6:187-195 (1987)).

[0187] The expression vectors listed herein are provided by way ofexample only of the well-known vectors available to those of ordinaryskill in the art that would be useful to express the nucleic acidmolecules. The person of ordinary skill in the art would be aware ofother vectors suitable for maintenance propagation or expression of thenucleic acid molecules described herein. These are found for example inSambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: ALaboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

[0188] The invention also encompasses vectors in which the nucleic acidsequences described herein are cloned into the vector in reverseorientation, but operably linked to a regulatory sequence that permitstranscription of antisense RNA. Thus, an antisense transcript can beproduced to all, or to a portion, of the nucleic acid molecule sequencesdescribed herein, including both coding and non-coding regions.Expression of this antisense RNA is subject to each of the parametersdescribed above in relation to expression of the sense RNA (regulatorysequences, constitutive or inducible expression, tissue-specificexpression).

[0189] The invention also relates to recombinant host cells containingthe vectors described herein. Host cells therefore include prokaryoticcells, lower eukaryotic cells such as yeast, other eukaryotic cells suchas insect cells, and higher eukaryotic cells such as mammalian cells.

[0190] The recombinant host cells are prepared by introducing the vectorconstructs described herein into the cells by techniques readilyavailable to the person of ordinary skill in the art. These include, butare not limited to, calcium phosphate transfection,DEAE-dextran-mediated transfection, cationic lipid-mediatedtransfection, electroporation, transduction, infection, lipofection, andother techniques such as those found in Sambrook, et al. (MolecularCloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

[0191] Host cells can contain more than one vector. Thus, differentnucleotide sequences can be introduced on different vectors of the samecell. Similarly, the nucleic acid molecules can be introduced eitheralone or with other nucleic acid molecules that are not related to thenucleic acid molecules such as those providing trans-acting factors forexpression vectors. When more than one vector is introduced into a cell,the vectors can be introduced independently, co-introduced or joined tothe nucleic acid molecule vector.

[0192] In the case of bacteriophage and viral vectors, these can beintroduced into cells as packaged or encapsulated virus by standardprocedures for infection and transduction. Viral vectors can bereplication-competent or replication-defective. In the case in whichviral replication is defective, replication will occur in host cellsproviding functions that complement the defects.

[0193] Vectors generally include selectable markers that enable theselection of the subpopulation of cells that contain the recombinantvector constructs. The marker can be contained in the same vector thatcontains the nucleic acid molecules described herein or may be on aseparate vector. Markers include tetracycline or ampicillin-resistancegenes for prokaryotic host cells and dihydrofolate reductase or neomycinresistance for eukaryotic host cells. However, any marker that providesselection for a phenotypic trait will be effective.

[0194] While the mature proteins can be produced in bacteria, yeast,mammalian cells, and other cells under the control of the appropriateregulatory sequences, cell-free transcription and translation systemscan also be used to produce these proteins using RNA derived from theDNA constructs described herein.

[0195] Where secretion of the peptide is desired, which is difficult toachieve with multi-transmembrane domain containing proteins such askinases, appropriate secretion signals are incorporated into the vector.The signal sequence can be endogenous to the peptides or heterologous tothese peptides.

[0196] Where the peptide is not secreted into the medium, which istypically the case with kinases, the protein can be isolated from thehost cell by standard disruption procedures, including freeze thaw,sonication, mechanical disruption, use of lysing agents and the like.The peptide can then be recovered and purified by well-knownpurification methods including ammonium sulfate precipitation, acidextraction, anion or cationic exchange chromatography, phosphocellulosechromatography, hydrophobic-interaction chromatography, affinitychromatography, hydroxylapatite chromatography, lectin chromatography,or high performance liquid chromatography.

[0197] It is also understood that depending upon the host cell inrecombinant production of the peptides described herein, the peptidescan have various glycosylation patterns, depending upon the cell, ormaybe non-glycosylated as when produced in bacteria. In addition, thepeptides may include an initial modified methionine in some cases as aresult of a host-mediated process.

[0198] Uses of Vectors and Host Cells

[0199] The recombinant host cells expressing the peptides describedherein have a variety of uses. First, the cells are useful for producinga secreted protein or peptide that can be further purified to producedesired amounts of secreted protein or fragments. Thus, host cellscontaining expression vectors are useful for peptide production.

[0200] Host cells are also useful for conducting cell-based assaysinvolving the secreted protein or secreted protein fragments, such asthose described above as well as other formats known in the art. Thus, arecombinant host cell expressing a native secreted protein is useful forassaying compounds that stimulate or inhibit secreted protein function.

[0201] Host cells are also useful for identifying secreted proteinmutants in which these functions are affected. If the mutants naturallyoccur and give rise to a pathology, host cells containing the mutationsare useful to assay compounds that have a desired effect on the mutantsecreted protein (for example, stimulating or inhibiting function) whichmay not be indicated by their effect on the native secreted protein.

[0202] Genetically engineered host cells can be further used to producenon-human transgenic animals. A transgenic animal is preferably amammal, for example a rodent, such as a rat or mouse, in which one ormore of the cells of the animal include a transgene. A transgene isexogenous DNA which is integrated into the genome of a cell from which atransgenic animal develops and which remains in the genome of the matureanimal in one or more cell types or tissues of the transgenic animal.These animals are useful for studying the function of a secreted proteinand identifying and evaluating modulators of secreted protein activity.Other examples of transgenic animals include non-human primates, sheep,dogs, cows, goats, chickens, and amphibians.

[0203] A transgenic animal can be produced by introducing nucleic acidinto the male pronuclei of a fertilized oocyte, e.g., by microinjection,retroviral infection, and allowing the oocyte to develop in apseudopregnant female foster animal. Any of the secreted proteinnucleotide sequences can be introduced as a transgene into the genome ofa non-human animal, such as a mouse.

[0204] Any of the regulatory or other sequences useful in expressionvectors can form part of the transgenic sequence. This includes intronicsequences and polyadenylation signals, if not already included. Atissue-specific regulatory sequence(s) can be operably linked to thetransgene to direct expression of the secreted protein to particularcells.

[0205] Methods for generating transgenic animals via embryo manipulationand microinjection, particularly animals such as mice, have becomeconventional in the art and are described, for example, in U.S. Pat.Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No.4,873,191 by Wagner et al. and in Hogan, B., Manipulating the MouseEmbryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1986). Similar methods are used for production of other transgenicanimals. A transgenic founder animal can be identified based upon thepresence of the transgene in its genome and/or expression of transgenicmRNA in tissues or cells of the animals. A transgenic founder animal canthen be used to breed additional animals carrying the transgene.Moreover, transgenic animals carrying a transgene can further be bred toother transgenic animals carrying other transgenes. A transgenic animalalso includes animals in which the entire animal or tissues in theanimal have been produced using the homologously recombinant host cellsdescribed herein.

[0206] In another embodiment, transgenic non-human animals can beproduced which contain selected systems that allow for regulatedexpression of the transgene. One example of such a system is thecre/loxP recombinase system of bacteriophage P1. For a description ofthe cre/loxP recombinase system, see, e.g., Lakso et al. PNAS89:6232-6236 (1992). Another example of a recombinase system is the FLPrecombinase system of S. cerevisiae (O'Gorman et al. Science251:1351-1355 (1991). If a cre/loxP recombinase system is used toregulate expression of the transgene, animals containing transgenesencoding both the Cre recombinase and a selected protein is required.Such animals can be provided through the construction of “double”transgenic animals, e.g., by mating two transgenic animals, onecontaining a transgene encoding a selected protein and the othercontaining a transgene encoding a recombinase.

[0207] Clones of the non-human transgenic animals described herein canalso be produced according to the methods described in Wilmut, I. et al.Nature 385:810-813 (1997) and PCT International Publication Nos. WO97/07668 and WO 97/07669. In brief, a cell, e.g., a somatic cell, fromthe transgenic animal can be isolated and induced to exit the growthcycle and enter G_(o) phase. The quiescent cell can then be fused, e.g.,through the use of electrical pulses, to an enucleated oocyte from ananimal of the same species from which the quiescent cell is isolated.The reconstructed oocyte is then cultured such that it develops tomorula or blastocyst and then transferred to pseudopregnant femalefoster animal. The offspring born of this female foster animal will be aclone of the animal from which the cell, e.g., the somatic cell, isisolated.

[0208] Transgenic animals containing recombinant cells that express thepeptides described herein are useful to conduct the assays describedherein in an in vivo context. Accordingly, the various physiologicalfactors that are present in vivo and that could effect substratebinding, secreted protein activation, and signal transduction, may notbe evident from in vitro cell-free or cell-based assays. Accordingly, itis useful to provide non-human transgenic animals to assay in vivosecreted protein function, including substrate interaction, the effectof specific mutant secreted proteins on secreted protein function andsubstrate interaction, and the effect of chimeric secreted proteins. Itis also possible to assess the effect of null mutations, that is,mutations that substantially or completely eliminate one or moresecreted protein functions.

[0209] All publications and patents mentioned in the above specificationare herein incorporated by reference. Various modifications andvariations of the described method and system of the invention will beapparent to those skilled in the art without departing from the scopeand spirit of the invention. Although the invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of theabove-described modes for carrying out the invention which are obviousto those skilled in the field of molecular biology or related fields areintended to be within the scope of the following claims.

1 4 1 1146 DNA Homo sapiens 1 atgagttgga ctgtgcctgt tgtgcgggccagccagagaa tgagctcggt gggagcgaat 60 ttcctatgcc tggggatggc cctgtgtctgcgtcaagcaa cgcgcatccc gctcaacggc 120 acctggctct tcacacccgt gagcaagatggcgactgtga agagtgagct tattgagcgt 180 ttcacttccg agaagcccgt tcatcacagtaaggtctcca tcataggaac tggatcggtg 240 ggcatggcct gcgctatcag catcttattaaaaggcttga gtgatgaact tgcccttgtg 300 gatcttgatg aagacaaact gaagggtgagacgatggatc ttcaacatgg cagccctttc 360 acgaaaatgc caaatattgt ttgtagcaaagattactttg tcacagcaaa ctccaaccta 420 gtgattatca cagcaggtgc acgccaagaaaagggagaaa cgcgccttaa tttagtccag 480 cgaaatgtgg ccatcttcaa gttaatgatttccagtattg tccagtacag cccccactgc 540 aaactgatta ttgtttccaa tccagtggatatcttaactt atgtagcttg gaagttgagt 600 gcatttccca aaaaccgtat tattggaagcggctgtaatc tggatactgc tcgttttcgt 660 ttcttgattg gacaaaagct tggtatccattctgaaagct gccatggatg gatcctcgga 720 gagcatggag actcaagtgt tcctgtgtggagtggagtga acatagctgg tgtccctttg 780 aaggatctga actctgatat aggaactgataaagatcctg agcaatggaa aaatgtccac 840 aaagaagtga ctgcaactgc ctatgagattattaaaatga aaggttatac ttcttgggcc 900 attggcctat ctgtggccga tttaacagaaagtattttga agaatcttag gagaatacat 960 ccagtttcca ccataactaa gggcctctatggaatagatg aagaagtatt cctcagtatt 1020 ccttgtatcc tgggagagaa cggtattaccaaccttataa agataaagct gacccctgaa 1080 gaagaggccc atctgaaaaa aagtgcaaaaacactctggg aaattcagaa taagcttaag 1140 ctttaa 1146 2 381 PRT Homo sapiens2 Met Ser Trp Thr Val Pro Val Val Arg Ala Ser Gln Arg Met Ser Ser 1 5 1015 Val Gly Ala Asn Phe Leu Cys Leu Gly Met Ala Leu Cys Leu Arg Gln 20 2530 Ala Thr Arg Ile Pro Leu Asn Gly Thr Trp Leu Phe Thr Pro Val Ser 35 4045 Lys Met Ala Thr Val Lys Ser Glu Leu Ile Glu Arg Phe Thr Ser Glu 50 5560 Lys Pro Val His His Ser Lys Val Ser Ile Ile Gly Thr Gly Ser Val 65 7075 80 Gly Met Ala Cys Ala Ile Ser Ile Leu Leu Lys Gly Leu Ser Asp Glu 8590 95 Leu Ala Leu Val Asp Leu Asp Glu Asp Lys Leu Lys Gly Glu Thr Met100 105 110 Asp Leu Gln His Gly Ser Pro Phe Thr Lys Met Pro Asn Ile ValCys 115 120 125 Ser Lys Asp Tyr Phe Val Thr Ala Asn Ser Asn Leu Val IleIle Thr 130 135 140 Ala Gly Ala Arg Gln Glu Lys Gly Glu Thr Arg Leu AsnLeu Val Gln 145 150 155 160 Arg Asn Val Ala Ile Phe Lys Leu Met Ile SerSer Ile Val Gln Tyr 165 170 175 Ser Pro His Cys Lys Leu Ile Ile Val SerAsn Pro Val Asp Ile Leu 180 185 190 Thr Tyr Val Ala Trp Lys Leu Ser AlaPhe Pro Lys Asn Arg Ile Ile 195 200 205 Gly Ser Gly Cys Asn Leu Asp ThrAla Arg Phe Arg Phe Leu Ile Gly 210 215 220 Gln Lys Leu Gly Ile His SerGlu Ser Cys His Gly Trp Ile Leu Gly 225 230 235 240 Glu His Gly Asp SerSer Val Pro Val Trp Ser Gly Val Asn Ile Ala 245 250 255 Gly Val Pro LeuLys Asp Leu Asn Ser Asp Ile Gly Thr Asp Lys Asp 260 265 270 Pro Glu GlnTrp Lys Asn Val His Lys Glu Val Thr Ala Thr Ala Tyr 275 280 285 Glu IleIle Lys Met Lys Gly Tyr Thr Ser Trp Ala Ile Gly Leu Ser 290 295 300 ValAla Asp Leu Thr Glu Ser Ile Leu Lys Asn Leu Arg Arg Ile His 305 310 315320 Pro Val Ser Thr Ile Thr Lys Gly Leu Tyr Gly Ile Asp Glu Glu Val 325330 335 Phe Leu Ser Ile Pro Cys Ile Leu Gly Glu Asn Gly Ile Thr Asn Leu340 345 350 Ile Lys Ile Lys Leu Thr Pro Glu Glu Glu Ala His Leu Lys LysSer 355 360 365 Ala Lys Thr Leu Trp Glu Ile Gln Asn Lys Leu Lys Leu 370375 380 3 3144 DNA Homo sapiens 3 cagaagtggt atcaaagttg caccaatctggccagaaact catgctcaga tactctgttt 60 aacaagagat cagctgggat gatggatgagaccaggggat ttccatagca ctaggaagtc 120 cccaagacca cgacctcaac ttaacttttttatcatcagc agccccacac agcctggcat 180 ccagtaggca cccaatactg atgggctttataagaacacc aaaagtgcac tctgggcctt 240 gatttatact accagtaggc atggggagacaaggaatgcc agtctgtgat tccagcaaga 300 ggcaagacga aaatccattt attcagcagctaatacatgt agcagggtga ttcattttcc 360 aaaaatatgc agtagtatgc tgagatttagtgctggagtg actcccagcc ggcccctacc 420 ctgctgaagc cccagccgac tgtcccagggtgggtcctgg ccaggactgg caccaaggca 480 gtggaatcct ttcaaaatag gcctccagcgccatagacca gaaagcaggc gcccaattct 540 gggacactgg gagatggtgc aggcagccaccagtatgcaa agcatagagc cctatccttc 600 ctggcagggt cacggttcag aaaacgtaaccttattttca tctgtagtta acagaagcat 660 gtttcctcag cattcatatt aaaaataaaaaactagtaat tccagggatc tcggttagcc 720 tcttaagcat gtcaactatt gataatactcttagcaaaac taactccaga aatcacttgc 780 tagagaagca gcccttcact gccttagtctgagcacccac ggaaagcacg tgttggagac 840 tctggcaagg cccgcgcagc cgcagtagagggccaggggg cggggcacgc gcacctgccg 900 tagagtgctg aaggtcctgc caacggctctcttggcgtct caacgttcgg atcagcagct 960 tttttccatt ctctctctcc acttcttcagtgagcagcca tgagttggac tgtgcctgtt 1020 gtgcgggcca gccagagaat gagctcggtgggagcgaatt tcctatgcct ggggatggcc 1080 ctgtgtctgc gtcaagcaac gcgcatcccgctcaacggca cctggctctt cacacccgtg 1140 agcaagatgg cgactgtgaa gagtgagcttattgagcgtt tcacttccga gaagcccgtt 1200 catcacagta aggtctccat cataggaactggatcggtgg gcatggcctg cgctatcagc 1260 atcttattaa aaggcttgag tgatgaacttgcccttgtgg atcttgatga agacaaactg 1320 aagggtgaga cgatggatct tcaacatggcagccctttca cgaaaatgcc aaatattgtt 1380 tgtagcaaag attactttgt cacagcaaactccaacctag tgattatcac agcaggtgca 1440 cgccaagaaa agggagaaac gcgccttaatttagtccagc gaaatgtggc catcttcaag 1500 ttaatgattt ccagtattgt ccagtacagcccccactgca aactgattat tgtttccaat 1560 ccagtggata tcttaactta tgtagcttggaagttgagtg catttcccaa aaaccgtatt 1620 attggaagcg gctgtaatct ggatactgctcgttttcgtt tcttgattgg acaaaagctt 1680 ggtatccatt ctgaaagctg ccatggatggatcctcggag agcatggaga ctcaagtgtt 1740 cctgtgtgga gtggagtgaa catagctggtgtccctttga aggatctgaa ctctgatata 1800 ggaactgata aagatcctga gcaatggaaaaatgtccaca aagaagtgac tgcaactgcc 1860 tatgagatta ttaaaatgaa aggttatacttcttgggcca ttggcctatc tgtggccgat 1920 ttaacagaaa gtattttgaa gaatcttaggagaatacatc cagtttccac cataactaag 1980 ggcctctatg gaatagatga agaagtattcctcagtattc cttgtatcct gggagagaac 2040 ggtattacca accttataaa gataaagctgacccctgaag aagaggccca tctgaaaaaa 2100 agtgcaaaaa cactctggga aattcagaataagcttaagc tttaaagttg cctaaaacta 2160 ccattccgaa attattgaag agatcatagatacaggatta tataacgaaa ttttgaataa 2220 acttgaattc ctaaaagatg gaaacaggaaagtaggtaga gtgattttcc tatttattta 2280 gtcctccagc tcttttattg agcatccacgtgctggacga tacttattta caattcctaa 2340 gtatttttgg tacctctgat gtagcagcacttgccatgtt atatatatgt agttggcatt 2400 tggttcccaa aaagtaggat gtaggtatttattgtgttct agaaattccg actcttttca 2460 ttagatatat gctatttctt tcattcttgctggtttatac ctatgttcat ttatatgctg 2520 taaaaaagta gtagcttctt ctacaatgtaaaaataaatg tacatacaaa aaaatgcagt 2580 agtatataca atcttttgtt ttgcttcctttgatagttaa taaattccgt ttgttgaatc 2640 aataaaatac ggcgagccat atgatttgcttcatgagaaa ccagatcaca aacccaaagg 2700 cttaaaacag atagacatta aaatggatattggccatacc ttcccagcat aatgatgaat 2760 gatgaagcct tggttccaac tgttgaagtgctcatgactc ccaatctgca tctgaagttt 2820 ctggagcagc gtctgatctg ccccctcacccaccgcatgc atcgtggcgc acacgtcatc 2880 caggatgctc atgatgccag gagggttctgatgggagcaa gaaggcaagg ccctagtcac 2940 tgacctctcc aaaacagaca atgcttatcaggtcctttct taggcaagga aacagctccc 3000 cagacttaag ataccatctt gaaagtcagtattccccttc aggcaagaat gcaattaata 3060 gtcccaaata gggagtcacc acttagatctaaagagggag gaaactccac tttccaaaaa 3120 gctgtttaac tccctgaagt ttta 3144 4331 PRT Mus musculus 4 Met Ala Thr Leu Lys Asp Gln Leu Ile Val Asn LeuLeu Lys Glu Glu 1 5 10 15 Gln Ala Pro Gln Asn Lys Ile Thr Val Val GlyVal Gly Ala Val Gly 20 25 30 Met Ala Cys Ala Ile Ser Ile Leu Met Lys AspLeu Ala Asp Glu Leu 35 40 45 Ala Leu Val Asp Val Met Glu Asp Lys Leu LysGly Glu Met Met Asp 50 55 60 Leu Gln His Gly Ser Leu Phe Leu Lys Thr ProLys Ile Val Ser Ser 65 70 75 80 Lys Asp Tyr Cys Val Thr Ala Asn Ser LysLeu Val Ile Ile Thr Ala 85 90 95 Gly Ala Arg Gln Gln Glu Gly Glu Ser ArgLeu Asn Leu Val Gln Arg 100 105 110 Asn Val Asn Ile Phe Lys Phe Ile IlePro Asn Ile Val Lys Tyr Ser 115 120 125 Pro His Cys Lys Leu Leu Ile ValSer Asn Pro Val Asp Ile Leu Thr 130 135 140 Tyr Val Ala Trp Lys Ile SerGly Phe Pro Lys Asn Arg Val Ile Gly 145 150 155 160 Ser Gly Cys Asn LeuAsp Ser Ala Arg Phe Arg Tyr Leu Met Gly Glu 165 170 175 Arg Leu Gly ValHis Ala Leu Ser Cys His Gly Trp Val Leu Gly Glu 180 185 190 His Gly AspSer Ser Val Pro Val Trp Ser Gly Val Asn Val Ala Gly 195 200 205 Val SerLeu Lys Ser Leu Asn Pro Glu Leu Gly Thr Asp Ala Asp Lys 210 215 220 GluGln Trp Lys Glu Val His Lys Gln Val Val Asp Ser Ala Tyr Glu 225 230 235240 Val Ile Lys Leu Lys Gly Tyr Thr Ser Trp Ala Ile Gly Leu Ser Val 245250 255 Ala Asp Leu Ala Glu Ser Ile Met Lys Asn Leu Arg Arg Val His Pro260 265 270 Ile Ser Thr Met Ile Lys Gly Leu Tyr Gly Ile Asn Glu Asp ValPhe 275 280 285 Leu Ser Val Pro Cys Ile Leu Gly Gln Asn Gly Ile Ser AspVal Val 290 295 300 Lys Val Thr Leu Thr Pro Glu Glu Glu Ala Arg Leu LysLys Ser Ala 305 310 315 320 Asp Thr Leu Trp Gly Ile Gln Lys Glu Leu Gln325 330

That which is claimed is:
 1. An isolated peptide consisting of an aminoacid sequence selected from the group consisting of: (a) an amino acidsequence shown in SEQ ID NO:2; (b) an amino acid sequence of an allelicvariant of an amino acid sequence shown in SEQ ID NO:2, wherein saidallelic variant is encoded by a nucleic acid molecule that hybridizesunder stringent conditions to the opposite strand of a nucleic acidmolecule shown in SEQ ID NOS:1 or 3; (c) an amino acid sequence of anortholog of an amino acid sequence shown in SEQ ID NO:2, wherein saidortholog is encoded by a nucleic acid molecule that hybridizes understringent conditions to the opposite strand of a nucleic acid moleculeshown in SEQ ID NOS:1 or 3; and (d) a fragment of an amino acid sequenceshown in SEQ ID NO:2, wherein said fragment comprises at least 10contiguous amino acids.
 2. An isolated peptide comprising an amino acidsequence selected from the group consisting of: (a) an amino acidsequence shown in SEQ ID NO:2; (b) an amino acid sequence of an allelicvariant of an amino acid sequence shown in SEQ ID NO:2, wherein saidallelic variant is encoded by a nucleic acid molecule that hybridizesunder stringent conditions to the opposite strand of a nucleic acidmolecule shown in SEQ ID NOS:1 or 3; (c) an amino acid sequence of anortholog of an amino acid sequence shown in SEQ ID NO:2, wherein saidortholog is encoded by a nucleic acid molecule that hybridizes understringent conditions to the opposite strand of a nucleic acid moleculeshown in SEQ ID NOS:1 or 3; and (d) a fragment of an amino acid sequenceshown in SEQ ID NO:2, wherein said fragment comprises at least 10contiguous amino acids.
 3. An isolated antibody that selectively bindsto a peptide of claim
 2. 4. An isolated nucleic acid molecule consistingof a nucleotide sequence selected from the group consisting of: (a) anucleotide sequence that encodes an amino acid sequence shown in SEQ IDNO:2; (b) a nucleotide sequence that encodes of an allelic variant of anamino acid sequence shown in SEQ ID NO:2, wherein said nucleotidesequence hybridizes under stringent conditions to the opposite strand ofa nucleic acid molecule shown in SEQ ID NOS:1 or 3; (c) a nucleotidesequence that encodes an ortholog of an amino acid sequence shown in SEQID NO:2, wherein said nucleotide sequence hybridizes under stringentconditions to the opposite strand of a nucleic acid molecule shown inSEQ ID NOS:1 or 3; (d) a nucleotide sequence that encodes a fragment ofan amino acid sequence shown in SEQ ID NO:2, wherein said fragmentcomprises at least 10 contiguous amino acids; and (e) a nucleotidesequence that is the complement of a nucleotide sequence of (a)-(d). 5.An isolated nucleic acid molecule comprising a nucleotide sequenceselected from the group consisting of: (a) a nucleotide sequence thatencodes an amino acid sequence shown in SEQ ID NO:2; (b) a nucleotidesequence that encodes of an allelic variant of an amino acid sequenceshown in SEQ ID NO:2, wherein said nucleotide sequence hybridizes understringent conditions to the opposite strand of a nucleic acid moleculeshown in SEQ ID NOS:1 or 3; (c) a nucleotide sequence that encodes anortholog of an amino acid sequence shown in SEQ ID NO:2, wherein saidnucleotide sequence hybridizes under stringent conditions to theopposite strand of a nucleic acid molecule shown in SEQ ID NOS:1 or 3;(d) a nucleotide sequence that encodes a fragment of an amino acidsequence shown in SEQ ID NO:2, wherein said fragment comprises at least10 contiguous amino acids; and (e) a nucleotide sequence that is thecomplement of a nucleotide sequence of (a)-(d).
 6. A gene chipcomprising a nucleic acid molecule of claim
 5. 7. A transgenic non-humananimal comprising a nucleic acid molecule of claim
 5. 8. A nucleic acidvector comprising a nucleic acid molecule of claim
 5. 9. A host cellcontaining the vector of claim
 8. 10. A method for producing any of thepeptides of claim 1 comprising introducing a nucleotide sequenceencoding any of the amino acid sequences in (a)-(d) into a host cell,and culturing the host cell under conditions in which the peptides areexpressed from the nucleotide sequence.
 11. A method for producing anyof the peptides of claim 2 comprising introducing a nucleotide sequenceencoding any of the amino acid sequences in (a)-(d) into a host cell,and culturing the host cell under conditions in which the peptides areexpressed from the nucleotide sequence.
 12. A method for detecting thepresence of any of the peptides of claim 2 in a sample, said methodcomprising contacting said sample with a detection agent thatspecifically allows detection of the presence of the peptide in thesample and then detecting the presence of the peptide.
 13. A method fordetecting the presence of a nucleic acid molecule of claim 5 in asample, said method comprising contacting the sample with anoligonucleotide that hybridizes to said nucleic acid molecule understringent conditions and determining whether the oligonucleotide bindsto said nucleic acid molecule in the sample.
 14. A method foridentifying a modulator of a peptide of claim 2, said method comprisingcontacting said peptide with an agent and determining if said agent hasmodulated the function or activity of said peptide.
 15. The method ofclaim 14, wherein said agent is administered to a host cell comprisingan expression vector that expresses said peptide.
 16. A method foridentifying an agent that binds to any of the peptides of claim 2, saidmethod comprising contacting the peptide with an agent and assaying thecontacted mixture to determine whether a complex is formed with theagent bound to the peptide.
 17. A pharmaceutical composition comprisingan agent identified by the method of claim 16 and a pharmaceuticallyacceptable carrier therefor.
 18. A method for treating a disease orcondition mediated by a human secreted protein, said method comprisingadministering to a patient a pharmaceutically effective amount of anagent identified by the method of claim
 16. 19. A method for identifyinga modulator of the expression of a peptide of claim 2, said methodcomprising contacting a cell expressing said peptide with an agent, anddetermining if said agent has modulated the expression of said peptide.20. An isolated human secreted peptide having an amino acid sequencethat shares at least 70% homology with an amino acid sequence shown inSEQ ID NO:2.
 21. A peptide according to claim 20 that shares at least 90percent homology with an amino acid sequence shown in SEQ ID NO:2. 22.An isolated nucleic acid molecule encoding a human secreted peptide,said nucleic acid molecule sharing at least 80 percent homology with anucleic acid molecule shown in SEQ ID NOS:1 or
 3. 23. A nucleic acidmolecule according to claim 22 that shares at least 90 percent homologywith a nucleic acid molecule shown in SEQ ID NOS:1 or 3.